Variants of Terminal Deoxynucleotidyl Transferase and Uses Thereof

ABSTRACT

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/242,904, filed Jan. 8, 2019, which application isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to variants of Terminal deoxynucleotidylTransferase (TdT) and uses thereof for the enzymatic synthesis ofnucleic acid sequences without template. More particularly, the presentinvention relates to such variants suitable to incorporate modifiednucleotides, for the synthesis of nucleic acid molecules with determinedor controlled sequences.

BACKGROUND

Methods for de novo chemical synthesis of nucleic acids based onsolid-phase phosphoramidite chemistry have been largely used and refinedover the past 40 years. The technique consists of a four-step chainelongation cycle that adds one base per cycle onto a growingoligonucleotide chain attached to a solid support matrix. Although ithas been the method of choice to synthesize nucleic acids during thepast decades, this technology has some notable limitations: It requiresthe use of multiple solvents and reagents, and due to limitations inchemical reaction efficiency, the length of synthetic oligonucleotidestypically do not exceed 150-200 bases. Moreover, these short fragmentsneed to be further assembled to provide the desired DNA sequence.

One alternative to chemical synthesis consists in using templateindependent DNA polymerases that will add reversible terminator modifiednucleotides to a growing single stranded chain of nucleic acids. Thisallows the addition of one type of nucleotide per cycle in a controlledfashion.

Some native enzymes are able to act on natural nucleotides in theabsence of template and so can catalyze the synthesis of nucleic acidsin an uncontrolled fashion. However, they are particularly inefficientto incorporate modified nucleotides and more particularly reversibleterminator modified nucleotides. Efforts have been made to develop newDNA polymerases able to act on modified nucleotides but the resultingenzymes are not fully satisfactory in terms of performances for thesynthesis of any type of nucleic acids.

So far, only few DNA polymerases that can act efficiently on singlestrand DNA (without the use of template) have been identified. The mostcharacterized polymerase having such template-independent activity isthe Terminal deoxynucleotidyl Transferase (TdT). TdT enzymes have beenextensively used to modify single stranded DNA for various types ofapplications including biotechnology, biomedical research and syntheticbiology. However, native TdT is poorly able to use modified nucleotides.

Several attempts to develop modified TdT with acceptable performance forthe incorporation of modified nucleotides have been carried over.However, the performances of the incorporation of such modifiednucleotides is still a limiting factor. Incorporation efficiency is thekey parameter driving the overall purity and yield of synthesis. Thesetwo characteristics of the synthesis process have a significant impactof quality, turnaround time and cost of nucleic acid products.

There is therefore a need to develop improved TdT capable to usemodified nucleotides in the absence of template, for developingefficient and cost-effective methods for the nucleic acid synthesis.

SUMMARY OF THE INVENTION

By working on TdT for de novo synthesis of polynucleotides withcontrolled sequence and without the use of a template, the inventorshave discovered that some targeted amino acid residues of the catalyticdomain of the TdT may be specifically modified to improve the ability ofsuch modified TdT for synthesizing polynucleotides. More particularly,the inventors have developed modified TdTs with targeted amino acidsubstitution(s) that lead to improve the enzymatic synthesis ofpolynucleotides and to reduce the overall cost of synthesizingpolynucleotides. In some embodiments, each of the modified TdTs presentsone or more targeted amino acids substitution as compared to wild-typeTdTs (such as SEQ ID NOs:1, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32 or 34) and N-terminal truncated versions thereof that comprise a TdTcatalytic domain. In some embodiments, each of the modified TdTs of theinvention possesses an amino acid sequence having a specified percentsequence identity with a catalytic domain of aTdT (such as SEQ ID NOs:2,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35) and having one ormore specified amino acid substitution(s). The template-independentpolymerases of the invention allow the enzymatic synthesis ofpolynucleotides at a faster rate, with less expense and higher quality.

It is therefore an object of the invention to provide variants ofTerminal deoxynucleotidyl Transferase (TdT) which (i) comprise an aminoacid sequence of a TdT catalytic domain or of a percent sequenceidentity of a TdT catalytic domain, such as set forth in SEQ ID NOs 2,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35, with at least anamino acid substitution at position corresponding to residue C302 (withrespect to the amino acid numbering of SEQ ID NO: 1), or functionallyequivalent residue, (ii) is capable of synthesizing a nucleic acidfragment without template and (iii) is capable of incorporating amodified nucleotide, such as a 3′-O-modified nucleotide onto a free3′-hydroxyl of a nucleic fragment.

More particularly, it is an object of the present invention to provideterminal deoxynucleotidyl transferase (TdT) variants comprising an aminoacid sequence at least 90% identical to SEQ ID NO: 2, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33 or 35 with a substitution at positioncorresponding to residue C173 with respect to SEQ ID NOs 2, 11, 13, 17,19, 21, 29 or 31, or at position corresponding to residue C172 withrespect to SEQ ID NO: 15, or at position corresponding to residue C178with respect to SEQ ID NO: 23, or at position corresponding to residueC174 with respect to SEQ ID NO: 25, or at position corresponding toresidue C171 with respect to SEQ ID NO: 27, or at position correspondingto residue C182 with respect to SEQ ID NO: 33, or at positioncorresponding to residue C176 with respect to SEQ ID NO: 35, wherein theTdT variant (i) is capable of synthesizing a nucleic acid fragmentwithout a template and (ii) is capable of incorporating a 3′-O-modifiednucleotide onto a free 3′-hydroxyl of a nucleic acid fragment. In someembodiments, the above percent identity value is at least 95 percentidentity with the indicated SEQ ID NOs; in some embodiments, the abovepercent identity value is at least 97 percent identity; in someembodiments, the above percent identity value is at least 98 percentidentity; in some embodiments, the above percent identity value is atleast 99 percent identity.

Advantageously, in regard to (iii), such 3′-O-modified nucleotide maycomprise a 3′-O—NH2-nucleoside triphosphate, a3′-O-azidomethyl-nucleoside triphosphate, a 3′-O-allyl-nucleosidetriphosphate, a 3′ 0-(2-nitrobenzyl)-nucleoside triphosphate, or a3′-O-propargyl-nucleoside triphosphate.

In a particular embodiment, the substitution is selected from:

C302G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:1; orC173G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:2; orC313G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO: 10; orC173G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:11; orC302G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:12; orC173G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO: 13; orC302G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:14; orC172G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:15; orC304G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:16; orC173G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:17; orC304G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:18; orC173G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:19; orC293G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:20; orC174G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:21; orC282G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:22; orC173G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:23; orC304G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:24; orC174G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:25; orC300G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:26; orC171G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:27; orC305G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:28; orC173G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:29; orC302G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:30; orC173G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:31; orC313G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:32; orC182G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:33; orC271G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:34; orC176G/R/P/A/V/S/N/Q/D with respect to SEQ ID NO:35.

In a further embodiment, the substitution is selected from:

C302G/R with respect to SEQ ID NO:1; or C302G/R with respect to SEQ IDNO:1; or C173G/R with respect to SEQ ID NO:2; or C302G/R with respect toSEQ ID NO:4; or C302G/R with respect to SEQ ID NO:9; or C313G/R withrespect to SEQ ID NO:10; or C173G/R with respect to SEQ ID NO:11; orC302G/R with respect to SEQ ID NO:12; or C173G/R with respect to SEQ IDNO:13; or C302G/R with respect to SEQ ID NO:14; or C172G/R with respectto SEQ ID NO:15; or C304G/R with respect to SEQ ID NO:16; or C173G/Rwith respect to SEQ ID NO:17; or C304G/R with respect to SEQ ID NO:18;or C173G/R with respect to SEQ ID NO:19; or C293G/R with respect to SEQID NO:20; or C173G/R with respect to SEQ ID NO:21; or C282G/R withrespect to SEQ ID NO:22; or C173G/R with respect to SEQ ID NO:23; orC304G/R with respect to SEQ ID NO:24; or C174G/R with respect to SEQ IDNO:25; or C300G/R with respect to SEQ ID NO:26; or C171G/R with respectto SEQ ID NO:27; or C305G/R with respect to SEQ ID NO:28; or C173G/Rwith respect to SEQ ID NO:29; or C302G/R with respect to SEQ ID NO:30;or C173G/R with respect to SEQ ID NO:31; or C313G/R with respect to SEQID NO:32; or C182G/R with respect to SEQ ID NO:33; or C271G/R withrespect to SEQ ID NO:34; or C176G/R with respect to SEQ ID NO:35.

In some embodiments, the invention is directed to compositionscomprising TdT variants comprising amino acid sequence having at least90 percent identity, or in some embodiments, at least 95 percentidentity, or in some embodiments, at least 97 percent identity, or insome embodiments, at least 98 percent identity, with a reference or wildtype TdT sequence selected from the group consisting of SEQ ID NOs: 2,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35, wherein (i) suchTdT variants have a mutation selected from C173G/R/P/A/V/S/N/Q/D, suchas C173G/R (wherein the amino acid residue number is with respect to SEQID NO: 2, or an equivalent residue number of SEQ ID NOs 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33 or 35) and (ii) such TdT variantsincorporate a modified nucleotide, such as a 3′-O-modified nucleosidetriphosphates, with greater efficiency, or at a higher rate, than thereference or wild type TdT.

In some embodiments, it is also an object of the invention to providetruncated variants of Terminal deoxynucleotidyl Transferase (TdT) eachof which (i) comprises an amino acid sequence with at least 95 percentidentity to any of SEQ ID NOs:11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33 or 35 with at least two amino acid substitutions, such as atleast three amino acid substitutions, selected from M192R/Q, L260P,C302G/R, R336L/N, D379V, R454P/N and E457N/L/T/S, (wherein residuenumbers are with respect to SEQ ID NO:1 or with respect to theirfunctionally equivalent residues numbers in SEQ ID NOs 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33 or 35), (ii) is able to synthesize anucleic acid fragment without a template and (iii) is able toincorporate a modified nucleotide into the nucleic acid fragment, forexample, a 3′-O-reversibly blocked deoxynucleoside triphosphate onto afree 3′-hydroxyl of a nucleic acid fragment. In further embodiments, theabove percent sequence identity value is at least 98 percent identitywith the specified sequences.

It is another object of the invention to provide a nucleic acid moleculeencoding a variant of a TdT as defined above and/or an expression vectorcomprising such nucleic acid molecule, and/or a host cell comprisingsuch nucleic acid molecule or expression vector.

It is a further object of the invention to provide a process forproducing a variant of TdT according to the invention, wherein a hostcell as defined above is cultivated under culture conditions allowingthe expression of the nucleic acid encoding said variant, and whereinthe variant is optionally retrieved.

The invention further relates to the use of a variant of TdT, forsynthesizing a nucleic acid molecule without template, by the successiveaddition of one or more 3′O-modified nucleotides to a nucleic acidfragment. In some embodiments, such methods comprise the steps of (a)providing an initiator comprising an oligonucleotide having a free3′-hydroxyl; (b) reacting under enzymatic extension conditions a TdTvariant of the invention with the initiator or an extended initiator inthe presence of a 3′-O-reversibly blocked nucleoside. In someembodiments, such method further includes steps of (c) deblocking theextended initiators to form extended initiators with free 3′-hydroxylsand (d) repeating steps (b) and (c) until a nucleic acid molecule of apredetermined sequence is synthesized.

It is also an object of the invention to provide a process forsynthesizing a nucleic acid molecule without template, comprising a stepof contacting a nucleic acid primer with both at least one nucleotide,such as at least one modified nucleotides, such as a 3′O-modifiednucleotide, and a variant of TdT according to the invention.

The present invention further provides a kit for performing a nucleotideincorporation reaction comprising a variant of TdT according to theinvention, and one or more nucleotides, such as one or more modifiednucleotides, such as a 3′O-modified nucleotides, and optionally at leastone nucleic acid primer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Purification assay of wild type (wt) TdT and different TdTvariants of the invention. Protein samples were loaded on SDS-PAGEanalysis gel and migrated through electrophoresis.

FIG. 2: Comparative results of performances for an elongation assayusing wt TdT and TdT variants of the invention. The assay involvesfluorescent labeled primers and 3′-O-amino reversible terminatormodified nucleotides. The results represent mean value of n=3experiments for each enzyme.

FIG. 3: Mass spectrum analysis of the results obtained for theelongation assay with different TdT variants of the invention. Only therelevant part of the mass spectrum is shown. The arrow shows the peak(mass) for the expected elongated primer.

DESCRIPTION OF THE INVENTION

The DNA polymerase families are divided into seven families based ontheir sequence homology and crystal structure. Among them, thepolymerases of PolX family represent a wide variety of polymerases fromreplicative polymerases to terminal transferase enzymes. Polymerasesfrom PolX family are present across a very wide range of eukaryoticorganisms. Polymerases from the PolX family are implicated in a vastvariety of biological processes and in particular in DNA damage repairmechanisms or error correction mechanisms. The PolX family regroupspolymerase β (Pol β), μ (Pol μ), λ (Pol λ), IV from yeast (Pol IV) andthe Terminal deoxynucleotidyl Transferase (TdT). TdT is naturallyimplicated in DNA repair and maintenance mechanisms. In particular, TdThas the unique ability to conserve a nucleotide polymerization activityeven in absence of template strand. In specific conditions and withnatural nucleotides, TdT is able to elongate DNA fragments with severalhundred nucleotides, in absence of any complementary strand. However,wild type TdT is totally unable to efficiently incorporatesugar-modified nucleotides.

It is thus the purpose of the present invention to provide variants ofTdT with targeted mutation(s) that allow them to incorporate modifiednucleotides into a nucleic fragment during synthesize of said nucleotidefragment. More particularly, the inventors have identified specificamino acid residues that may be advantageously substituted, alone or incombination, to improve the ability of the enzyme to synthesize nucleicacid fragments of various length and with pre-determined sequence,including by using modified nucleotides.

Definitions

As used therein, the terms “mutant” and “variant” may be usedinterchangeably to refer to polypeptides related to or derived from SEQID NOs:2, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 2627, 28, 29, 30, 31, 32, 33, 34 or 35 and comprising a modification or analteration, i.e., a substitution, insertion, and/or deletion, at one ormore (e.g., several) positions and having both a polymerase activitywithout template and ability to incorporate 3′-O-modified nucleosidetriphosphates into a nucleic acid chain. The variants may be obtained byvarious techniques well known in the art. In particular, examples oftechniques for altering the DNA sequence encoding the wild-type protein,include, but are not limited to, site-directed mutagenesis, randommutagenesis and synthetic oligonucleotide construction. Mutagenesisactivities consist in deleting, inserting or substituting one or severalamino-acids in the sequence of a protein or in the case of the inventionof a polymerase. Targeted amino-acids could be concomitant ordistributed along the whole sequence of the polymerase. Specific motifsor structural features could be targeted for example.

The terms “modification” or “alteration” as used herein in relation to aposition or amino acid mean that the amino acid in the specific positionhas been modified compared to the amino acid of the wild-type protein.

A “substitution” means that an amino acid residue is replaced by anotheramino acid residue. For example, the term “substitution” refers to thereplacement of an amino acid residue by another selected from thenaturally-occurring standard 20 amino acid residues, rare naturallyoccurring amino acid residues (e.g. hydroxyproline, hydroxylysine,allohydroxylysine, 6-N-methylysine, N-ethylglycine, N-methylglycine,N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline,pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), andnon-naturally occurring amino acid residue, often made synthetically,(e.g. cyclohexyl-alanine). For example, the term “substitution” refersto the replacement of an amino acid residue by another selected from thenaturally-occurring standard 20 amino acid residues. The sign “+”indicates a combination of substitutions.

The amino acids are herein represented by their one-letter orthree-letters code according to the following nomenclature: A: alanine(Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid(Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I:isoleucine (Ile); K: lysine (Lys); L: leucine (Leu); M: methionine(Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R:arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W:tryptophan (Trp) and Y: tyrosine (Tyr).

In the present document, the following terminology is used to designatea substitution: L238A denotes that amino acid residue (Leucine, L) atposition 238 of the parent sequence is changed to an Alanine (A).A132V/I/M denotes that amino acid residue (Alanine, A) at position 132of the parent sequence is substituted by one of the following aminoacids: Valine (V), Isoleucine (I), or Methionine (M). The substitutioncan be a conservative or non-conservative substitution. Examples ofconservative substitutions are within the groups of basic amino acids(arginine, lysine and histidine), acidic amino acids (glutamic acid andaspartic acid), polar amino acids (glutamine, asparagine and threonine),hydrophobic amino acids (methionine, leucine, isoleucine, cysteine andvaline), aromatic amino acids (phenylalanine, tryptophan and tyrosine),and small amino acids (glycine, alanine and serine).

As used herein, the terms “sequence identity” or “identity” refer to thenumber (or fraction expressed as a percentage %) of matches (identicalamino acid residues) between two polypeptide sequences. The sequenceidentity is determined by comparing the sequences when aligned so as tomaximize overlap and identity while minimizing sequence gaps. Inparticular, sequence identity may be determined using any of a number ofmathematical global or local alignment algorithms, depending on thelength of the two sequences. Sequences of similar lengths are alignedusing a global alignment algorithm (e.g. Needleman and Wunsch algorithm;Needleman and Wunsch, 1970) which aligns the sequences optimally overthe entire length, while sequences of substantially different lengthsare aligned using a local alignment algorithm (e.g. Smith and Watermanalgorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul etal., 1997; Altschul et al., 2005)). Alignment for purposes ofdetermining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software available on internet web sitessuch as http://blast.ncbi.nlm.nih.gov/orhttp://www.ebi.ac.uk/Tools/emboss/. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithm needed to achieve maximal alignment over the full length ofthe sequences being compared. For purposes herein, % amino acid sequenceidentity values refer to values generated using the pair wise sequencealignment program EMBOSS Needle, that creates an optimal globalalignment of two sequences using the Needleman-Wunsch algorithm, whereinall search parameters are set to default values, i.e. Scoringmatrix=BLOSUM62, Gap open=10, Gap extend=0.5, End gap penalty=false, Endgap open=10 and End gap extend=0.5.

Herein, the terms “peptide”, “polypeptide”, “protein”, “enzyme”, referto a chain of amino acids linked by peptide bonds, regardless of thenumber of amino acids forming said chain.

Unless otherwise specified, the positions disclosed in the presentapplication are numbered by reference to the amino acid sequence setforth in a specified SEQ ID NO.

Variants of TdT

The present invention provides variants of TdT enzyme that can be usedfor synthesizing polynucleotides of predetermined sequences, such as DNAor RNA, without the use of template strand. The TdT variants of theinvention allow modified nucleotides, and more particularly 3′O-modifiednucleotides, to be used in an enzyme-mediated method of polynucleotidesynthesis, such as described by Hiatt et al, U.S. Pat. No. 5,763,594.

In some embodiments of the invention, “modified Terminaldesoxyribonucleotidyl Transferase”, “modified TdT”, “variants ofTerminal desoxyribonucleotidyl Transferase” and “variants of TdT” referto enzymes that comprise an amino acid segment that shares at least 80%identity with an amino acid sequence of one of the amino acid sequencesset forth in SEQ ID NOs:2, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33 or 35, excepting at least one amino acid residue substitution. Insome embodiments, the variant of TdT comprises an amino acid sequencethat shares at least 90% identity with SEQ ID NOs:2, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, and having at least one amino acidresidue substitution. In still other embodiments, the variant of TdTcomprises an amino acid sequence that shares at least 95% identity withSEQ ID NOs:2, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, andhaving at least one amino acid residue substitution. In still otherembodiments, the variant of TdT comprises an amino acid sequence thatshares at least 98% identity with SEQ ID NOs:2, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, and having at least one amino acid residuesubstitution.

In some cases, variants of the present invention may be describedaccording to their mutations on specific residues, whose positions aredetermined by alignment with or reference to the enzymatic sequence SEQID NO:1 or SEQ ID NO:2, which corresponds to the amino acid sequences ofmurine TdT and truncated murine TdT respectively. The variants of theinvention may also be described directly with reference to SEQ IDnumbers of corresponding reference sequences.

By “functionally equivalent residue” is meant a residue in a sequence ofa TdT of sequence homologous to SEQ ID NO:1 or to SEQ ID NO:2 and havingan identical functional role. Functionally equivalent residues areidentified by using sequence alignments, for example, using the Mutalinline alignment software(http://multalin.toulouse.inra.fr/multalin/multalin.html; 1988, Nucl.Acids Res., 16 (22), 10881-10890). After alignment, the functionallyequivalent residues are at homologous positions on the differentsequences considered. Sequence alignments and identification offunctionally equivalent residues may be between any TdT and theirnatural variants, including inter-species.

TdT can be found in many organisms or microorganisms. All those TdT aregood candidates for performing the present invention. In particular,modifications to alter a particular TdT sequence to give said polymerasean increased ability to incorporate modified nucleotides, can target anyother TdT sequence. Accordingly, mutations or combinations describedherein by reference to SEQ ID NO:1, and more particularly to SEQ ID NO:2that corresponds to amino acid residues 130 to 510 of SEQ ID NO:1, canbe transposed to any other TdT sequence.

In some embodiments, the invention comprises a variant of Terminaldeoxynucleotidyl Transferase (TdT) that (i) comprises an amino acidsequence having at least 80%, such as at least 85%, 90%, 95% or 99%identity with an amino acid sequence selected from SEQ ID NO: 2, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35, with at least an aminoacid substitution at position corresponding to a functionally equivalentresidue of residue C173 with respect to SEQ ID NO:11, (ii) is able tosynthesize a nucleic acid fragment without template and (iii) is able toincorporate a modified nucleoside triphosphate, such as a 3′-O-blockednucleoside triphosphate, into the nucleic fragment.

Indeed, the inventors have discovered that such substitution has a greatimpact on both surface and interaction properties of the enzyme withnucleotides, which may allow incorporation of 3′O-modified nucleotidesin a nucleic acid sequence.

Further embodiments of TdT variants of the invention are listed asentries in Tables 1A through 1C (single substitutions), Tables 2Athrough 2C (two substitutions), Tables 3A through 3C (threesubstitutions), and Tables 4A through 4F (four substitutions), whereineach such variant TdT is defined by the indicated SEQ ID NO in therighthand column modified by the substitution(s) listed in the lefthandcolumn of the same row as the SEQ ID NO. A “non-wild type” substitutionmeans that the substitution may be any amino acid except for the aminoacid at the indicated position in the wild type sequence, orequivalently, the sequence of the indicated SEQ ID NO.

TABLE 1A TdT variants at position C173 (SEQ ID NO: 2) or functionallyequivalent positions of the indicated SEQ ID NO Non-wild typesubstitution at SEQ ID NO C173 2 C313 10 C173 11 C302 12 C173 13 C302 14C172 15 C304 16 C173 17 C304 18 C173 19 C293 20 C173 21 C282 22 C178 23C304 24 C174 25 C300 26 C171 27 C305 28 C173 29 C302 30 C173 31 C313 32C182 33 C271 34 C176 35

TABLE 1B Further TdT variants at position C173 (SEQ ID NO: 2) orfunctionally equivalent positions of the indicated SEQ ID NOSubstitution SEQ ID NO C173/G/R/P/A/V/S/N/Q/D 2 C313/G/R/P/A/V/S/N/Q/D10 C173/G/R/P/A/V/S/N/Q/D 11 C302/G/R/P/A/V/S/N/Q/D 12C173/G/R/P/A/V/S/N/Q/D 13 C302/G/R/P/A/V/S/N/Q/D 14C172/G/R/P/A/V/S/N/Q/D 15 C304/G/R/P/A/V/S/N/Q/D 16C173/G/R/P/A/V/S/N/Q/D 17 C304/G/R/P/A/V/S/N/Q/D 18C173/G/R/P/A/V/S/N/Q/D 19 C293/G/R/P/A/V/S/N/Q/D 20C173/G/R/P/A/V/S/N/Q/D 21 C282/G/R/P/A/V/S/N/Q/D 22C178/G/R/P/A/V/S/N/Q/D 23 C304/G/R/P/A/V/S/N/Q/D 24C174/G/R/P/A/V/S/N/Q/D 25 C300/G/R/P/A/V/S/N/Q/D 26C171/G/R/P/A/V/S/N/Q/D 27 C305/G/R/P/A/V/S/N/Q/D 28C173/G/R/P/A/V/S/N/Q/D 29 C302/G/R/P/A/V/S/N/Q/D 30C173/G/R/P/A/V/S/N/Q/D 31 C313/G/R/P/A/V/S/N/Q/D 32C182/G/R/P/A/V/S/N/Q/D 33 C271/G/R/P/A/V/S/N/Q/D 34C176/G/R/P/A/V/S/N/Q/D 35

TABLE 1C Further TdT variants at position C173 (SEQ ID NO: 2) orfunctionally equivalent positions of the indicated SEQ ID NOSubstitutions SEQ ID NO C173/G/R 2 C313/G/R 10 C173/G/R 11 C302/G/R 12C173/G/R 13 C302/G/R 14 C172/G/R 15 C304/G/R 16 C173/G/R 17 C304/G/R 18C173/G/R 19 C293/G/R 20 C173/G/R 21 C282/G/R 22 C178/G/R 23 C304/G/R 24C174/G/R 25 C300/G/R 26 C171/G/R 27 C305/G/R 28 C173/G/R 29 C302/G/R 30C173/G/R 31 C313/G/R 32 C182/G/R 33 C271/G/R 34 C176/G/R 35

TABLE 2A Further TdT variants at position C173 (SEQ ID NO: 2) andposition M63 (SEQ ID NO: 2) or functionally equivalent positions of theindicated SEQ ID NO Non-wildtype substitutions at locations SEQ ID NOM63 + C173 2 M63 + C173 11 M63 + C173 13 L62 + C172 15 M63 + C173 17M63 + C173 19 R64 + C173 21 M73 + C178 23 M64 + C174 25 M61 + C171 27M63 + C173 29 L63 + C173 31 M63 + C182 33 M66 + C176 35

TABLE 2B Further TdT variants at position C173 (SEQ ID NO: 2) andposition M63 (SEQ ID NO: 2) or functionally equivalent positions of theindicated SEQ ID NO Substitutions and substitution positions SEQ ID NOM63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D 2 M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D 11 M63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D 13L62R/Q/G/A/V/D/N/H/E + C172G/R/P/A/V/S/N/Q/D 15 M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D 17 M63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D 19R64R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D 21 M73R/Q/G/A/V/D/N/H/E +C178G/R/P/A/V/S/N/Q/D 23 M64R/Q/G/A/V/D/N/H/E + C174G/R/P/A/V/S/N/Q/D 25M61R/Q/G/A/V/D/N/H/E + C171G/R/P/A/V/S/N/Q/D 27 M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D 29 L63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D 31M63R/Q/G/A/V/D/N/H/E + C182G/R/P/A/V/S/N/Q/D 33 M66R/Q/G/A/V/D/N/H/E +C176G/R/P/A/V/S/N/Q/D 35

TABLE 2C Further TdT variants at position C173 (SEQ ID NO: 2) andposition M63 (SEQ ID NO: 2) or functionally equivalent positions of theindicated SEQ ID NO Substitutions and substitution positions SEQ ID NOM63R/Q + C173G/R 2 M63R/Q + C173G/R 11 M63R/Q + C173G/R 13 L62R/Q +C172G/R 15 M63R/Q + C173G/R 17 M63R/Q + C173G/R 19 R64R/Q + C173G/R 21M73R/Q + C178G/R 23 M64R/Q + C174G/R 25 M61R/Q + C171G/R 27 M63R/Q +C173G/R 29 L63R/Q + C173G/R 31 M63R/Q + C182G/R 33 M66R/Q + C176G/R 35

TABLE 3A Further TdT variants at positions C173 (SEQ ID NO: 2), M63 (SEQID NO: 2) and R207 (SEQ ID NO: 2) or functionally equivalent positionsof the indicated SEQ ID NO Mutations SEQ ID NO M63 + C173 + R207 2 M63 +C173 + R207 11 M63 + C173 + R207 13 L62 + C172 + R206 15 M63 + C173 +R207 17 M63 + C173 + R207 19 R64 + C173 + R208 21 M73 + C178 + R207 23M64 + C174 + R208 25 M61 + C171 + R205 27 M63 + C173 + R207 29 L63 +C173 + R207 31 M63 + C182 + R216 33 M66 + C176 + R210 35

TABLE 3B Further TdT variants at positions C173 (SEQ ID NO: 2), M63 (SEQID NO: 2) and R207 (SEQ ID NO: 2) or functionally equivalent positionsof the indicated SEQ ID NO Mutations SEQ ID NO M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 2 R207N/L/K/H/G/D/A/P M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 11 R207 N/L/K/H/G/D/A/P M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 13 R207 N/L/K/H/G/D/A/P L62R/Q/G/A/V/D/N/H/E +C172G/R/P/A/V/S/N/Q/D + 15 R206 N/L/K/H/G/D/A/P M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 17 R207 N/L/K/H/G/D/A/P M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 19 R207 N/L/K/H/G/D/A/P R64Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 21 R208 N/L/K/H/G/D/A/P M73R/Q/G/A/V/D/N/H/E +C178G/R/P/A/V/S/N/Q/D + 23 R207 N/L/K/H/G/D/A/P M64R/Q/G/A/V/D/N/H/E +C174G/R/P/A/V/S/N/Q/D + 25 R208 N/L/K/H/G/D/A/P M61R/Q/G/A/V/D/N/H/E +C171G/R/P/A/V/S/N/Q/D + 27 R205 N/L/K/H/G/D/A/P M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 29 R207 N/L/K/H/G/D/A/P L63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 31 R207N/L/K/H/G/D/A/P M63R/Q/G/A/V/D/N/H/E +C182G/R/P/A/V/S/N/Q/D 2 + 33 R216N/L/K/H/G/D/A/P M66R/Q/G/A/V/D/N/H/E +C176G/R/P/A/V/S/N/Q/D + 35 R210N/L/K/H/G/D/A/P

TABLE 3C Further TdT variants at positions C173 (SEQ ID NO: 2), M63 (SEQID NO: 2) and R207 (SEQ ID NO: 2) or functionally equivalent positionsof the indicated SEQ ID NO Mutations SEQ ID NO M63R/Q + C173G/R +R207L/N 2 M63R/Q + C173G/R + R207L/N 11 M63R/Q + C173G/R + R207L/N 13M62R/Q + C172G/R + R206L/N 15 M63R/Q + C173G/R + R207L/N 17 M63R/Q +C173G/R + R207L/N 19 R64Q + C173G/R + R208L/N 21 M73R/Q + C178G/R +R207N/L 23 M64R/Q + C174G/R + R208 N/L 25 M61R/Q + C171G/R + R205N/L 27M63R/Q + C173G/R + R207L/N 29 L63R/Q + C173G/R + R207L/N 31 M63R/Q +C182G/R + R216N/L 33 M66R/Q + C176G/R + R210N/L 35

TABLE 4A Further TdT variants at positions C173 (SEQ ID NO: 2), M63 (SEQID NO: 2), R207 (SEQ ID NO: 2) and R325 (SEQ ID NO: 2) or functionallyequivalent positions of the indicated SEQ ID NO Mutations SEQ ID NOM63 + C173 + R207 + R325 2 M63 + C173 + R207 + R324 11 M63 + C173 +R207 + R324 13 L62 + C172 + R206 + R320 15 M63 + C173 + R207 + R331 17M63 + C173 + R207 + P325 19 R64 + C173 + R208 + T331 21 M73 + C178 +R207 + R325 23 M64 + C174 + R208 + P326 25 M61 + C171 + R205 + R323 27M63 + C173 + R207 + R328 29 L63 + C173 + R207 + R325 31 M63 + C182 +R216 + R338 33 M66 + C176 + R210 + R328 35

TABLE 4B Further TdT variants at positions C173 (SEQ ID NO: 2), M63 (SEQID NO: 2), R207 (SEQ ID NO: 2) and R325 (SEQ ID NO: 2) or functionallyequivalent positions of the indicated SEQ ID NO Mutations SEQ ID NOM63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 2 R207N/L/K/H/G/D/A/P +R325P/N/A/L/K/H/G/D M63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 11R207 N/L/K/H/G/D/A/P + R324P/N/A/L/K/H/G/D M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 13 R207 N/L/K/H/G/D/A/P + R324P/N/A/L/K/H/G/DL62R/Q/G/A/V/D/N/H/E + C172G/R/P/A/V/S/N/Q/D + 15 R206 N/L/K/H/G/D/A/P +R320P/N/A/L/K/H/G/D M63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 17R207 N/L/K/H/G/D/A/P + R331P/N/A/L/K/H/G/D M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 19 R207 N/L/K/H/G/D/A/P + P325N/A/L/K/H/G/DR64Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 21 R208 N/L/K/H/G/D/A/P +T331P/N/A/L/K/H/G/D M73R/Q/G/A/V/D/N/H/E + C178G/R/P/A/V/S/N/Q/D + 23R207 N/L/K/H/G/D/A/P + R325P/N/A/L/K/H/G/D M64R/Q/G/A/V/D/N/H/E +C174G/R/P/A/V/S/N/Q/D + 25 R208 N/L/K/H/G/D/A/P + P326N/A/L/K/H/G/DM61R/Q/G/A/V/D/N/H/E + C171G/R/P/A/V/S/N/Q/D + 27 R205 N/L/K/H/G/D/A/P +R323P/N/A/L/K/H/G/D M63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 29R207 N/L/K/H/G/D/A/P + R328P/N/A/L/K/H/G/D L63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 31 R207N/L/K/H/G/D/A/P + R325P/N/A/L/K/H/G/DM63R/Q/G/A/V/D/N/H/E + C182G/R/P/A/V/S/N/Q/D + 33 R216N/L/K/H/G/D/A/P +R338P/N/A/L/K/H/G/D M66R/Q/G/A/V/D/N/H/E + C176G/R/P/A/V/S/N/Q/D + 35R210N/L/K/H/G/D/A/P + R328P/N/A/L/K/H/G/D

TABLE 4C Further TdT variants at positions C173 (SEQ ID NO: 2), M63 (SEQID NO: 2), R207 (SEQ ID NO: 2) and R325 (SEQ ID NO: 2) or functionallyequivalent positions of the indicated SEQ ID NO Mutations SEQ ID NOM63R/Q + C173G/R + R207N/L + R325P/N 2 M63R/Q + C173G/R + R207N/L +R324P/N 11 M63R/Q + C173G/R + R207N/L + R324P/N 13 L62R/Q + C172G/R +R206N/L + R320P/N 15 M63R/Q + C173G/R + R207N/L + R331P/N 17 M63R/Q +C173G/R + R207N/L + P325N 19 R64Q/G + C173G/R + R208N/L + T331P/N 21M73R/Q/G + C178G/R + R207N/L + R325P/N 23 M64R/Q + C174G/R + R208N/L +P326N 25 M61R/Q + C171G/R + R205N/L + R323P/N 27 M63R/Q + C173G/R +R207N/L + R328P/N 29 L63R/Q + C173G/R + R207N/L + R325P/N 31 M63R/Q +C182G/R + R216N/L + R338P/N 33 M66R/Q + C176G/R + R210N/L + R328P/N 35

TABLE 4D Further TdT variants at positions C173 (SEQ ID NO: 2), M63 (SEQID NO: 2), R207 (SEQ ID NO: 2) and E328 (SEQ ID NO: 2) or functionallyequivalent positions of the indicated SEQ ID NO Mutations SEQ ID NOM63 + C173 + R207 + E328 2 M63 + C173 + R207 + E327 11 M63 + C173 +R207 + E327 13 L62 + C172 + R206 + G323 15 M63 + C173 + R207 + E334 17M63 + C173 + R207 + E327 19 R64 + C173 + R208 + E334 21 M73 + C178 +R207 + E328 23 M64 + C174 + R208 + E329 25 M61 + C171 + R205 + E326 27M63 + C173 + R207 + E331 29 L63 + C173 + R207 + E328 31 M63 + C182 +R216 + E341 33 M66 + C176 + R210 + E331 35

TABLE 4E Further TdT variants at positions C173 (SEQ ID NO: 2), M63 (SEQID NO: 2), R207 (SEQ ID NO: 2) and E328 (SEQ ID NO: 2) or functionallyequivalent positions of the indicated SEQ ID NO Mutations SEQ ID NOM63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 2 R207N/L/K/H/G/D/A/P +E328N/L/T/S M63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 11 R207N/L/K/H/G/D/A/P + E327N/L/T/S M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 13 R207 N/L/K/H/G/D/A/P + E327N/L/T/SL62R/Q/G/A/V/D/N/H/E + C172G/R/P/A/V/S/N/Q/D + 15 R206 N/L/K/H/G/D/A/P +G323N/L/T/S M63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 17 R207N/L/K/H/G/D/A/P + E334N/L/T/S M63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 19 R207 N/L/K/H/G/D/A/P + E327N/L/T/SR64Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 21 R208 N/L/K/H/G/D/A/P +E334N/L/T/S M73R/Q/G/A/V/D/N/H/E + C178G/R/P/A/V/S/N/Q/D + 23 R207N/L/K/H/G/D/A/P + E328N/L/T/S M64R/Q/G/A/V/D/N/H/E +C174G/R/P/A/V/S/N/Q/D + 25 R208 N/L/K/H/G/D/A/P + E329N/L/T/SM61R/Q/G/A/V/D/N/H/E + C171G/R/P/A/V/S/N/Q/D + 27 R205 N/L/K/H/G/D/A/P +E326N/L/T/S M63R/Q/G/A/V/D/N/H/E + C173G/R/P/A/V/S/N/Q/D + 29 R207N/L/K/H/G/D/A/P + E331N/L/T/S L63R/Q/G/A/V/D/N/H/E +C173G/R/P/A/V/S/N/Q/D + 31 R207N/L/K/H/G/D/A/P + E328N/L/T/SM63R/Q/G/A/V/D/N/H/E + C182G/R/P/A/V/S/N/Q/D + 33 R216N/L/K/H/G/D/A/P +E341N/L/T/S M66R/Q/G/A/V/D/N/H/E + C176G/R/P/A/V/S/N/Q/D + 35R210N/L/K/H/G/D/A/P + E331N/L/T/S

TABLE 4F Further TdT variants at positions C173 (SEQ ID NO: 2), M63 (SEQID NO: 2), R207 (SEQ ID NO: 2) and E328 (SEQ ID NO: 2) or functionallyequivalent positions of the indicated SEQ ID NO Mutations SEQ ID NOM63R/Q + C173G/R + R207N/L + E328N/L/T/S 2 M63R/Q + C173G/R + R207 N/L +E327N/L/T/S 11 M63R/Q + C173G/R + R207N/L + E327N/L/T/S 13 L62R/Q +C172G/R + R206N/L + G323N/L/T/S 15 M63R/Q + C173G/R + R207N/L +E334N/L/T/S 17 M63R/Q + C173G/R + R207N/L + E327N/L/T/S 19 R64Q/G +C173G/R + R208N/L + E334N/L/T/S 21 M73R/Q + C178G/R + R207N/L +E328N/L/T/S 23 M64R/Q + C174G/R + R208N/L + E329N/L/T/S 25 M61R/Q +C171G/R + R205N/L + E326N/L/T/S 27 M63R/Q/G + C173G/R + R207N/L +E331N/L/T/S 29 L63R/Q + C173G/R + R207N/L + E328N/L/T/S 31 M63R/Q +C182G/R + R216N/L + E341N/L/T/S 33 M66R/Q + C176G/R + R210N/L +E331N/L/T/S 35

Advantageously, the substitution is selected from CzzzG/R/P/A/V/S/N/Q/D,where Czzz represents an amino acid residue number functionallyequivalent to C173 of SEQ ID NO:2 in SEQ ID NOs:11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33 and 35, respectively, and such as from CzzzG/R,where Czzz represents an amino acid residue number functionallyequivalent to C173 of SEQ ID NO: 2 in SEQ ID NOs:11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33 and 35, respectively.

In a particular embodiment, the variant further comprises at least oneamino acid substitution at position corresponding to functionallyequivalent residues of residues selected from M63, R207, R324 and E327,of SEQ ID NO:11.

According to the invention, all variants of TdT as disclosed above areable to both synthesize a nucleic acid fragment without template andincorporate a modified nucleotide into the nucleic acid fragment.Advantageously, said variants have an increased ability to incorporate amodified nucleotide, such as a 3′O-modified nucleotide, into a nucleicacid fragment as compared to a TdT of SEQ ID NOs:2, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33 and 35.

In some of the embodiments described above, the efficiency of a variantTdT in incorporating a 3′O-modified nucleoside triphosphate is at least110 percent that of a wild type TdT of sequence SEQ ID NO:2, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33 and 35 in other embodiments, theefficiency of a variant TdT in incorporating a 3′O-modified nucleosidetriphosphate is at least 150 percent that of a wild type TdT of sequenceSEQ ID NOs:2, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35; inother embodiments, the efficiency of a variant TdT in incorporating a3′O-modified nucleoside triphosphate is at least 200 percent that of awild type TdT of sequence SEQ ID NOs:2, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33 and 35.

The present invention further provides a variant of TdT having the aminoacid sequence as set forth in SEQ ID NO:2 or functionally equivalentsequence, with at least one substitution or combination of substitutionsas listed in Table 5 or Table 6. The variants of the invention compriseat least the amino acid substitutions listed in the left column andcalled “Variable Mutations”, or functionally equivalent residues, andoptionally one or both combination of substitutions listed in the rightcolumn and called “Optional Constant Mutations”, or functionallyequivalent sequence.

TABLE 5 Variants of TdT having the amino acid sequence of SEQ ID NO: 2or an amino acid sequence within a specified percent sequence identitythereof, with at least a substitution on residue C173 and other residuesas indicated (wherein the amino acid position numbers are with respectto SEQ ID NO: 2). Name Variable Mutations Optional Constant MutationsDS1 M63R + L131P + C173R + R207L + D250V + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS2 M63R + L131P + C173R + R207L +D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS3 M63R + L131P + C173R + R207L + D250V + R325P L52F + A108V +R354K and/or G284L/S + H287D + E289A DS4 M63R + L131P + C173R + R207L +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS5 M63R + L131P + C173R + R207L + D250V + R325A + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS6 M63R + L131P + C173R +R207L + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS7 M63R + L131P + C173R + R207L + D250V + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS8 M63R + L131P + C173R + R207L +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS9M63R + L131P + C173R + R207L + D250V L52F + A108V + R354K and/orG284L/S + H287D + E289A DS10 M63R + L131P + C173R + R207L + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS11 M63R +L131P + C173R + R207L + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS12 M63R + L131P + C173R + R207L + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS13 M63R + L131P + C173R +R207L + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS14 M63R + L131P + C173R + R207L + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS15 M63R + L131P + C173R + R207L +R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS16 M63R +L131P + C173R + R207L + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS17 M63R + L131P + C173R + R207L + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS18 M63R + L131P + C173R + R207LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS19 M63R + L131P +C173R + R207N + D250V + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS20 M63R + L131P + C173R + R207N + D250V +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS21M63R + L131P + C173R + R207N + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS22 M63R + L131P + C173R + R207N + D250V +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS23M63R + L131P + C173R + R207N + D250V + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS24 M63R + L131P + C173R + R207N +D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS25M63R + L131P + C173R + R207N + D250V + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS26 M63R + L131P + C173R + R207N + D250V +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS27 M63R +L131P + C173R + R207N + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS28 M63R + L131P + C173R + R207N + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS29 M63R + L131P + C173R +R207N + R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS30 M63R + L131P + C173R + R207N + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS31 M63R + L131P + C173R + R207N +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS32M63R + L131P + C173R + R207N + R325A + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS33 M63R + L131P + C173R + R207N + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS34 M63R + L131P + C173R +R207N + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS35M63R + L131P + C173R + R207N + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS36 M63R + L131P + C173R + R207N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS37 M63R + L131P + C173R + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS38M63R + L131P + C173R + D250V + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS39 M63R + L131P + C173R + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS40 M63R + L131P + C173R +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS41 M63R + L131P + C173R + D250V + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS42 M63R + L131P + C173R + D250V +R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS43 M63R +L131P + C173R + D250V + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS44 M63R + L131P + C173R + D250V + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS45 M63R + L131P + C173R + D250VL52F + A108V + R354K and/or G284L/S + H287D + E289A DS46 M63R + L131P +C173R + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS47 M63R + L131P + C173R + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS48 M63R + L131P + C173R + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS49 M63R + L131P + C173R +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS50M63R + L131P + C173R + R325A + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS51 M63R + L131P + C173R + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS52 M63R + L131P + C173R + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS53 M63R + L131P +C173R + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS54M63R + L131P + C173R L52F + A108V + R354K and/or G284L/S + H287D + E289ADS55 M63R + L131P + C173G + R207L + D250V + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS56 M63R + L131P + C173G + R207L +D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS57 M63R + L131P + C173G + R207L + D250V + R325P L52F + A108V +R354K and/or G284L/S + H287D + E289A DS58 M63R + L131P + C173G + R207L +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS59 M63R + L131P + C173G + R207L + D250V + R325A + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS60 M63R + L131P + C173G +R207L + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS61 M63R + L131P + C173G + R207L + D250V + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS62 M63R + L131P + C173G + R207L +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS63M63R + L131P + C173G + R207L + D250V L52F + A108V + R354K and/orG284L/S + H287D + E289A DS64 M63R + L131P + C173G + R207L + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS65 M63R +L131P + C173G + R207L + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS66 M63R + L131P + C173G + R207L + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS67 M63R + L131P + C173G +R207L + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS68 M63R + L131P + C173G + R207L + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS69 M63R + L131P + C173G + R207L +R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS70 M63R +L131P + C173G + R207L + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS71 M63R + L131P + C173G + R207L + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS72 M63R + L131P + C173G + R207LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS73 M63R + L131P +C173G + R207N + D250V + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS74 M63R + L131P + C173G + R207N + D250V +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS75M63R + L131P + C173G + R207N + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS76 M63R + L131P + C173G + R207N + D250V +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS77M63R + L131P + C173G + R207N + D250V + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS78 M63R + L131P + C173G + R207N +D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS79M63R + L131P + C173G + R207N + D250V + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS80 M63R + L131P + C173G + R207N + D250V +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS81 M63R +L131P + C173G + R207N + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS82 M63R + L131P + C173G + R207N + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS83 M63R + L131P + C173G +R207N + R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS84 M63R + L131P + C173G + R207N + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS85 M63R + L131P + C173G + R207N +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS86M63R + L131P + C173G + R207N + R325A + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS87 M63R + L131P + C173G + R207N + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS88 M63R + L131P + C173G +R207N + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS89M63R + L131P + C173G + R207N + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS90 M63R + L131P + C173G + R207N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS91 M63R + L131P + C173G + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS92M63R + L131P + C173G + D250V + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS93 M63R + L131P + C173G + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS94 M63R + L131P + C173G +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS95 M63R + L131P + C173G + D250V + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS96 M63R + L131P + C173G + D250V +R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS97 M63R +L131P + C173G + D250V + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS98 M63R + L131P + C173G + D250V + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS99 M63R + L131P + C173G + D250VL52F + A108V + R354K and/or G284L/S + H287D + E289A DS100 M63R + L131P +C173G + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS101 M63R + L131P + C173G + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS102 M63R + L131P + C173G + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS103 M63R + L131P +C173G + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS104 M63R + L131P + C173G + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS105 M63R + L131P + C173G + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS106 M63R + L131P +C173G + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS107M63R + L131P + C173G + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS108 M63R + L131P + C173G L52F + A108V + R354K and/orG284L/S + H287D + E289A DS163 M63R + C173R + R207L + D250V + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS164 M63R +C173R + R207L + D250V + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS165 M63R + C173R + R207L + D250V + R325PL52F + A108V + R354K and/or G284L/S + H287D + E289A DS166 M63R + C173R +R207L + D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS167 M63R + C173R + R207L + D250V + R325A + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS168 M63R + C173R +R207L + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS169 M63R + C173R + R207L + D250V + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS170 M63R + C173R + R207L + D250V +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS171 M63R +C173R + R207L + D250V L52F + A108V + R354K and/or G284L/S + H287D +E289A DS172 M63R + C173R + R207L + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS173 M63R + C173R + R207L + R325P +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS174 M63R +C173R + R207L + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS175 M63R + C173R + R207L + R325A + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS176 M63R + C173R + R207L + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS177 M63R +C173R + R207L + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS178 M63R + C173R + R207L + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS179 M63R + C173R + R207L + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS180 M63R + C173R + R207LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS52 M63R + C173R +R207N + D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS182 M63R + C173R + R207N + D250V + R325P + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS183 M63R + C173R +R207N + D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS184 M63R + C173R + R207N + D250V + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS185 M63R + C173R + R207N +D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS186 M63R + C173R + R207N + D250V + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS187 M63R + C173R + R207N + D250V +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS188 M63R +C173R + R207N + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS189 M63R + C173R + R207N + D250V L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS190 M63R + C173R + R207N + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS191 M63R +C173R + R207N + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS63 M63R + C173R + R207N + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS193 M63R + C173R + R207N + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS194 M63R +C173R + R207N + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS195 M63R + C173R + R207N + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS196 M63R + C173R + R207N + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS197 M63R + C173R +R207N + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS198M63R + C173R + R207N L52F + A108V + R354K and/or G284L/S + H287D + E289ADS199 M63R + C173R + D250V + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS200 M63R + C173R + D250V + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS201 M63R + C173R +D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS202M63R + C173R + D250V + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS203 M63R + C173R + D250V + R325A + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS204 M63R + C173R +D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS205M63R + C173R + D250V + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS206 M63R + C173R + D250V + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS207 M63R + C173R + D250V L52F + A108V +R354K and/or G284L/S + H287D + E289A DS208 M63R + C173R + R325P + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS209 M63R + C173R +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS210M63R + C173R + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289ADS211 M63R + C173R + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS212 M63R + C173R + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS213 M63R + C173R + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS214 M63R + C173R + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS215 M63R + C173R + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS216 M63R + C173RL52F + A108V + R354K and/or G284L/S + H287D + E289A DS217 M63R + C173G +R207L + D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS218 M63R + C173G + R207L + D250V + R325P + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS219 M63R + C173G +R207L + D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS220 M63R + C173G + R207L + D250V + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS221 M63R + C173G + R207L +D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS222 M63R + C173G + R207L + D250V + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS223 M63R + C173G + R207L + D250V +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS224 M63R +C173G + R207L + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS225 M63R + C173G + R207L + D250V L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS226 M63R + C173G + R207L + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS227 M63R +C173G + R207L + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS228 M63R + C173G + R207L + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS229 M63R + C173G + R207L + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS230 M63R +C173G + R207L + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS231 M63R + C173G + R207L + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS232 M63R + C173G + R207L + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS233 M63R + C173G +R207L + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS234M63R + C173G + R207L L52F + A108V + R354K and/or G284L/S + H287D + E289ADS235 M63R + C173G + R207N + D250V + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS236 M63R + C173G + R207N + D250V +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS108M63R + C173G + R207N + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS238 M63R + C173G + R207N + D250V + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS239 M63R +C173G + R207N + D250V + R325A + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS240 M63R + C173G + R207N + D250V + R325AL52F + A108V + R354K and/or G284L/S + H287D + E289A DS241 M63R + C173G +R207N + D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS242 M63R + C173G + R207N + D250V + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS243 M63R + C173G + R207N + D250V L52F +A108V + R354K and/or G284L/S + H287D + E289A DS244 M63R + C173G +R207N + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS245 M63R + C173G + R207N + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS246 M63R + C173G + R207N + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS247 M63R + C173G +R207N + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS248 M63R + C173G + R207N + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS249 M63R + C173G + R207N + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS250 M63R + C173G +R207N + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS251M63R + C173G + R207N + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS252 M63R + C173G + R207N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS253 M63R + C173G + D250V + R325P + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS254 M63R + C173G +D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS255 M63R + C173G + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS256 M63R + C173G + D250V + R325A + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS257 M63R + C173G +D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS258 M63R + C173G + D250V + R325A L52F + A108V + R354K and/orG284L/S + H287D + E289A DS259 M63R + C173G + D250V + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS131 M63R + C173G +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS261M63R + C173G + D250V L52F + A108V + R354K and/or G284L/S + H287D + E289ADS262 M63R + C173G + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS263 M63R + C173G + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS264 M63R + C173G + R325P L52F + A108V +R354K and/or G284L/S + H287D + E289A DS265 M63R + C173G + R325A + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS266 M63R + C173G +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS267M63R + C173G + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289ADS268 M63R + C173G + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS269 M63R + C173G + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS270 M63R + C173G L52F + A108V + R354K and/or G284L/S +H287D + E289A DS325 M63Q + L131P + C173R + R207L + D250V + R325P + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS326 M63Q + L131P +C173R + R207L + D250V + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS327 M63Q + L131P + C173R + R207L + D250V +R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS328 M63Q +L131P + C173R + R207L + D250V + R325A + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS329 M63Q + L131P + C173R + R207L +D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS330 M63Q + L131P + C173R + R207L + D250V + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS331 M63Q + L131P + C173R +R207L + D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS332 M63Q + L131P + C173R + R207L + D250V + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS333 M63Q + L131P + C173R +R207L + D250V L52F + A108V + R354K and/or G284L/S + H287D + E289A DS334M63Q + L131P + C173R + R207L + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS335 M63Q + L131P + C173R + R207L + R325P +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS207 M63Q +L131P + C173R + R207L + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS337 M63Q + L131P + C173R + R207L + R325A + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS338 M63Q + L131P +C173R + R207L + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS339 M63Q + L131P + C173R + R207L + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS340 M63Q + L131P + C173R +R207L + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS341M63Q + L131P + C173R + R207L + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS342 M63Q + L131P + C173R + R207L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS343 M63Q + L131P +C173R + R207N + D250V + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS344 M63Q + L131P + C173R + R207N + D250V +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS345M63Q + L131P + C173R + R207N + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS346 M63Q + L131P + C173R + R207N + D250V +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS347M63Q + L131P + C173R + R207N + D250V + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS348 M63Q + L131P + C173R +R207N + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS349 M63Q + L131P + C173R + R207N + D250V + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS350 M63Q + L131P + C173R +R207N + D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS351 M63Q + L131P + C173R + R207N + D250V L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS352 M63Q + L131P + C173R + R207N +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS353M63Q + L131P + C173R + R207N + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS354 M63Q + L131P + C173R + R207N + R325PL52F + A108V + R354K and/or G284L/S + H287D + E289A DS355 M63Q + L131P +C173R + R207N + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS356 M63Q + L131P + C173R + R207N + R325A + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS357 M63Q + L131P +C173R + R207N + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS358 M63Q + L131P + C173R + R207N + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS359 M63Q + L131P + C173R + R207N +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS360 M63Q +L131P + C173R + R207N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS361 M63Q + L131P + C173R + D250V + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS362 M63Q + L131P + C173R +D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS363 M63Q + L131P + C173R + D250V + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS364 M63Q + L131P + C173R + D250V +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS365M63Q + L131P + C173R + D250V + R325A + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS366 M63Q + L131P + C173R + D250V + R325AL52F + A108V + R354K and/or G284L/S + H287D + E289A DS367 M63Q + L131P +C173R + D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS368 M63Q + L131P + C173R + D250V + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS369 M63Q + L131P + C173R + D250V L52F +A108V + R354K and/or G284L/S + H287D + E289A DS370 M63Q + L131P +C173R + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS371 M63Q + L131P + C173R + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS372 M63Q + L131P + C173R + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS373 M63Q + L131P +C173R + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS374 M63Q + L131P + C173R + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS375 M63Q + L131P + C173R + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS376 M63Q + L131P +C173R + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS377M63Q + L131P + C173R + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS378 M63Q + L131P + C173R L52F + A108V + R354K and/orG284L/S + H287D + E289A DS250 M63Q + L131P + C173G + R207L + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS380M63Q + L131P + C173G + R207L + D250V + R325P + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS381 M63Q + L131P + C173G +R207L + D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS382 M63Q + L131P + C173G + R207L + D250V + R325A + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS383 M63Q + L131P +C173G + R207L + D250V + R325A + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS384 M63Q + L131P + C173G + R207L + D250V +R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS385 M63Q +L131P + C173G + R207L + D250V + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS386 M63Q + L131P + C173G + R207L + D250V +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS387 M63Q +L131P + C173G + R207L + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS388 M63Q + L131P + C173G + R207L + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS389 M63Q + L131P +C173G + R207L + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS390 M63Q + L131P + C173G + R207L + R325P L52F + A108V +R354K and/or G284L/S + H287D + E289A DS391 M63Q + L131P + C173G +R207L + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS392 M63Q + L131P + C173G + R207L + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS393 M63Q + L131P + C173G +R207L + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS394M63Q + L131P + C173G + R207L + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS395 M63Q + L131P + C173G + R207L + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS396 M63Q + L131P +C173G + R207L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS397M63Q + L131P + C173G + R207N + D250V + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS398 M63Q + L131P + C173G +R207N + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS399 M63Q + L131P + C173G + R207N + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS400 M63Q + L131P +C173G + R207N + D250V + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS401 M63Q + L131P + C173G + R207N + D250V +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS402M63Q + L131P + C173G + R207N + D250V + R325A L52F + A108V + R354K and/orG284L/S + H287D + E289A DS403 M63Q + L131P + C173G + R207N + D250V +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS404 M63Q +L131P + C173G + R207N + D250V + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS405 M63Q + L131P + C173G + R207N + D250VL52F + A108V + R354K and/or G284L/S + H287D + E289A DS406 M63Q + L131P +C173G + R207N + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS407 M63Q + L131P + C173G + R207N + R325P + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS408 M63Q + L131P +C173G + R207N + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS409 M63Q + L131P + C173G + R207N + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS410 M63Q + L131P + C173G +R207N + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS411 M63Q + L131P + C173G + R207N + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS412 M63Q + L131P + C173G + R207N +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS284 M63Q +L131P + C173G + R207N + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS414 M63Q + L131P + C173G + R207N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS415 M63Q + L131P + C173G + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS287M63Q + L131P + C173G + D250V + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS417 M63Q + L131P + C173G + D250V + R325PL52F + A108V + R354K and/or G284L/S + H287D + E289A DS289 M63Q + L131P +C173G + D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS419 M63Q + L131P + C173G + D250V + R325A + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS420 M63Q + L131P +C173G + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS421 M63Q + L131P + C173G + D250V + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS422 M63Q + L131P + C173G + D250V +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS423 M63Q +L131P + C173G + D250V L52F + A108V + R354K and/or G284L/S + H287D +E289A DS424 M63Q + L131P + C173G + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS425 M63Q + L131P + C173G + R325P +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS426 M63Q +L131P + C173G + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS427 M63Q + L131P + C173G + R325A + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS428 M63Q + L131P + C173G + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS429 M63Q +L131P + C173G + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS430 M63Q + L131P + C173G + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS431 M63Q + L131P + C173G + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS432 M63Q + L131P + C173GL52F + A108V + R354K and/or G284L/S + H287D + E289A DS487 M63Q + C173R +R207L + D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS488 M63Q + C173R + R207L + D250V + R325P + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS489 M63Q + C173R +R207L + D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS490 M63Q + C173R + R207L + D250V + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS491 M63Q + C173R + R207L +D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS492 M63Q + C173R + R207L + D250V + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS493 M63Q + C173R + R207L + D250V +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS494 M63Q +C173R + R207L + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS495 M63Q + C173R + R207L + D250V L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS496 M63Q + C173R + R207L + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS497 M63Q +C173R + R207L + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS498 M63Q + C173R + R207L + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS499 M63Q + C173R + R207L + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS500 M63Q +C173R + R207L + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS501 M63Q + C173R + R207L + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS502 M63Q + C173R + R207L + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS503 M63Q + C173R +R207L + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS504M63Q + C173R + R207L L52F + A108V + R354K and/or G284L/S + H287D + E289ADS505 M63Q + C173R + R207N + D250V + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS506 M63Q + C173R + R207N + D250V +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS507M63Q + C173R + R207N + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS508 M63Q + C173R + R207N + D250V + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS509 M63Q +C173R + R207N + D250V + R325A + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS510 M63Q + C173R + R207N + D250V + R325AL52F + A108V + R354K and/or G284L/S + H287D + E289A DS511 M63Q + C173R +R207N + D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS512 M63Q + C173R + R207N + D250V + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS513 M63Q + C173R + R207N + D250V L52F +A108V + R354K and/or G284L/S + H287D + E289A DS514 M63Q + C173R +R207N + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS515 M63Q + C173R + R207N + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS516 M63Q + C173R + R207N + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS517 M63Q + C173R +R207N + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS518 M63Q + C173R + R207N + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS519 M63Q + C173R + R207N + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS520 M63Q + C173R +R207N + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS521M63Q + C173R + R207N + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS522 M63Q + C173R + R207N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS523 M63Q + C173R + D250V + R325P + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS524 M63Q + C173R +D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS525 M63Q + C173R + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS526 M63Q + C173R + D250V + R325A + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS527 M63Q + C173R +D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS528 M63Q + C173R + D250V + R325A L52F + A108V + R354K and/orG284L/S + H287D + E289A DS529 M63Q + C173R + D250V + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS530 M63Q + C173R +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS531M63Q + C173R + D250V L52F + A108V + R354K and/or G284L/S + H287D + E289ADS532 M63Q + C173R + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS533 M63Q + C173R + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS534 M63Q + C173R + R325P L52F + A108V +R354K and/or G284L/S + H287D + E289A DS535 M63Q + C173R + R325A + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS536 M63Q + C173R +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS537M63Q + C173R + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289ADS538 M63Q + C173R + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS539 M63Q + C173R + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS540 M63Q + C173R L52F + A108V + R354K and/or G284L/S +H287D + E289A DS541 M63Q + C173G + R207L + D250V + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS542 M63Q + C173G +R207L + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS543 M63Q + C173G + R207L + D250V + R325P L52F + A108V +R354K and/or G284L/S + H287D + E289A DS544 M63Q + C173G + R207L +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS545 M63Q + C173G + R207L + D250V + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS546 M63Q + C173G + R207L +D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS547M63Q + C173G + R207L + D250V + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS548 M63Q + C173G + R207L + D250V + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS549 M63Q + C173G +R207L + D250V L52F + A108V + R354K and/or G284L/S + H287D + E289A DS550M63Q + C173G + R207L + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS551 M63Q + C173G + R207L + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS552 M63Q + C173G +R207L + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS553M63Q + C173G + R207L + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS554 M63Q + C173G + R207L + R325A + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS555 M63Q + C173G +R207L + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS556M63Q + C173G + R207L + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS557 M63Q + C173G + R207L + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS558 M63Q + C173G + R207L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS559 M63Q + C173G + R207N +D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS560 M63Q + C173G + R207N + D250V + R325P + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS561 M63Q + C173G + R207N +D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS562M63Q + C173G + R207N + D250V + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS563 M63Q + C173G + R207N + D250V + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS564 M63Q +C173G + R207N + D250V + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS565 M63Q + C173G + R207N + D250V + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS566 M63Q + C173G + R207N +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS567M63Q + C173G + R207N + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS568 M63Q + C173G + R207N + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS569 M63Q + C173G + R207N +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS570M63Q + C173G + R207N + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS571 M63Q + C173G + R207N + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS572 M63Q + C173G + R207N +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS573M63Q + C173G + R207N + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS574 M63Q + C173G + R207N + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS575 M63Q + C173G + R207N + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS576 M63Q + C173G + R207NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS577 M63Q + C173G +D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS578 M63Q + C173G + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS579 M63Q + C173G + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS580 M63Q + C173G +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS581 M63Q + C173G + D250V + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS582 M63Q + C173G + D250V + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS583 M63Q + C173G +D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS584M63Q + C173G + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS585 M63Q + C173G + D250V L52F + A108V + R354K and/orG284L/S + H287D + E289A DS586 M63Q + C173G + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS587 M63Q + C173G +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS588M63Q + C173G + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289ADS589 M63Q + C173G + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS590 M63Q + C173G + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS591 M63Q + C173G + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS592 M63Q + C173G + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS593 M63Q + C173G + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS594 M63Q + C173GL52F + A108V + R354K and/or G284L/S + H287D + E289A DS649 L131P +C173R + R207L + D250V + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS650 L131P + C173R + R207L + D250V + R325P +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS651 L131P +C173R + R207L + D250V + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS652 L131P + C173R + R207L + D250V + R325A + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS653 L131P + C173R +R207L + D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS654 L131P + C173R + R207L + D250V + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS655 L131P + C173R + R207L +D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS656L131P + C173R + R207L + D250V + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS657 L131P + C173R + R207L + D250V L52F +A108V + R354K and/or G284L/S + H287D + E289A DS658 L131P + C173R +R207L + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS659 L131P + C173R + R207L + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS660 L131P + C173R + R207L + R325PL52F + A108V + R354K and/or G284L/S + H287D + E289A DS661 L131P +C173R + R207L + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS662 L131P + C173R + R207L + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS663 L131P + C173R + R207L + R325AL52F + A108V + R354K and/or G284L/S + H287D + E289A DS664 L131P +C173R + R207L + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS665 L131P + C173R + R207L + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS666 L131P + C173R + R207L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS667 L131P + C173R + R207N + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS668L131P + C173R + R207N + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS669 L131P + C173R + R207N + D250V +R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS670 L131P +C173R + R207N + D250V + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS671 L131P + C173R + R207N + D250V + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS672 L131P +C173R + R207N + D250V + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS673 L131P + C173R + R207N + D250V + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS674 L131P + C173R + R207N +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS675L131P + C173R + R207N + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS676 L131P + C173R + R207N + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS677 L131P + C173R + R207N +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS678L131P + C173R + R207N + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS679 L131P + C173R + R207N + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS680 L131P + C173R + R207N +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS681L131P + C173R + R207N + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS682 L131P + C173R + R207N + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS683 L131P + C173R + R207N + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS684 L131P +C173R + R207N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS685L131P + C173R + D250V + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS686 L131P + C173R + D250V + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS687 L131P +C173R + D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS688 L131P + C173R + D250V + R325A + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS689 L131P + C173R + D250V + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS690 L131P +C173R + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS691 L131P + C173R + D250V + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS692 L131P + C173R + D250V + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS693 L131P + C173R + D250VL52F + A108V + R354K and/or G284L/S + H287D + E289A DS694 L131P +C173R + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS695 L131P + C173R + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS696 L131P + C173R + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS697 L131P + C173R + R325A + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS698 L131P +C173R + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS699 L131P + C173R + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS700 L131P + C173R + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS701 L131P + C173R + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS702 L131P + C173R L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS703 L131P + C173G + R207L + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS704L131P + C173G + R207L + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS705 L131P + C173G + R207L + D250V +R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS706 L131P +C173G + R207L + D250V + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS707 L131P + C173G + R207L + D250V + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS708 L131P +C173G + R207L + D250V + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS709 L131P + C173G + R207L + D250V + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS710 L131P + C173G + R207L +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS711L131P + C173G + R207L + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS712 L131P + C173G + R207L + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS713 L131P + C173G + R207L +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS714L131P + C173G + R207L + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS715 L131P + C173G + R207L + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS716 L131P + C173G + R207L +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS717L131P + C173G + R207L + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS718 L131P + C173G + R207L + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS719 L131P + C173G + R207L + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS720 L131P +C173G + R207L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS721L131P + C173G + R207N + D250V + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS722 L131P + C173G + R207N + D250V +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS723L131P + C173G + R207N + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS724 L131P + C173G + R207N + D250V + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS725 L131P +C173G + R207N + D250V + R325A + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS726 L131P + C173G + R207N + D250V + R325AL52F + A108V + R354K and/or G284L/S + H287D + E289A DS727 L131P +C173G + R207N + D250V + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS728 L131P + C173G + R207N + D250V + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS729 L131P + C173G + R207N + D250VL52F + A108V + R354K and/or G284L/S + H287D + E289A DS730 L131P +C173G + R207N + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS731 L131P + C173G + R207N + R325P + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS732 L131P + C173G + R207N + R325PL52F + A108V + R354K and/or G284L/S + H287D + E289A DS733 L131P +C173G + R207N + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS734 L131P + C173G + R207N + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS735 L131P + C173G + R207N + R325AL52F + A108V + R354K and/or G284L/S + H287D + E289A DS736 L131P +C173G + R207N + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS737 L131P + C173G + R207N + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS738 L131P + C173G + R207N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS739 L131P + C173G + D250V + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS740 L131P +C173G + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS741 L131P + C173G + D250V + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS742 L131P + C173G + D250V + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS743 L131P +C173G + D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS744 L131P + C173G + D250V + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS745 L131P + C173G + D250V + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS746 L131P +C173G + D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS747 L131P + C173G + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS748 L131P + C173G + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS749 L131P + C173G + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS750 L131P +C173G + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS751L131P + C173G + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS752 L131P + C173G + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS753 L131P + C173G + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS754 L131P + C173G + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS755 L131P +C173G + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS756L131P + C173G L52F + A108V + R354K and/or G284L/S + H287D + E289A DS811C173R + R207L + D250V + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS812 C173R + R207L + D250V + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS813 C173R +R207L + D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS814 C173R + R207L + D250V + R325A + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS815 C173R + R207L + D250V + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS816 C173R +R207L + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS817 C173R + R207L + D250V + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS818 C173R + R207L + D250V + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS819 C173R + R207L + D250VL52F + A108V + R354K and/or G284L/S + H287D + E289A DS820 C173R +R207L + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS821 C173R + R207L + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS822 C173R + R207L + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS823 C173R + R207L + R325A + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS824 C173R +R207L + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS825 C173R + R207L + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS826 C173R + R207L + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS827 C173R + R207L + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS828 C173R + R207L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS829 C173R + R207N + D250V + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS830 C173R +R207N + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS831 C173R + R207N + D250V + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS832 C173R + R207N + D250V + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS833 C173R +R207N + D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS834 C173R + R207N + D250V + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS835 C173R + R207N + D250V + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS836 C173R +R207N + D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS837 C173R + R207N + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS838 C173R + R207N + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS839 C173R + R207N + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS840 C173R +R207N + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS841C173R + R207N + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS842 C173R + R207N + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS843 C173R + R207N + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS844 C173R + R207N + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS845 C173R +R207N + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS846C173R + R207N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS847C173R + D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS848 C173R + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS849 C173R + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS850 C173R + D250V +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS851C173R + D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS852 C173R + D250V + R325A L52F + A108V + R354K and/orG284L/S + H287D + E289A DS853 C173R + D250V + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS854 C173R + D250V + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS855 C173R + D250V L52F +A108V + R354K and/or G284L/S + H287D + E289A DS856 C173R + R325P + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS857 C173R +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS858C173R + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS859C173R + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS860 C173R + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS861 C173R + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS862 C173R + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS863 C173R + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS864 C173R L52F + A108V + R354K and/or G284L/S + H287D +E289A DS865 C173G + R207L + D250V + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS866 C173G + R207L + D250V + R325P +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS867 C173G +R207L + D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS868 C173G + R207L + D250V + R325A + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS869 C173G + R207L + D250V + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS870 C173G +R207L + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS871 C173G + R207L + D250V + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS872 C173G + R207L + D250V + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS873 C173G + R207L + D250VL52F + A108V + R354K and/or G284L/S + H287D + E289A DS874 C173G +R207L + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS875 C173G + R207L + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS876 C173G + R207L + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS877 C173G + R207L + R325A + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS878 C173G +R207L + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS879 C173G + R207L + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS880 C173G + R207L + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS881 C173G + R207L + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS882 C173G + R207L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS883 C173G + R207N + D250V + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS884 C173G +R207N + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS885 C173G + R207N + D250V + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS886 C173G + R207N + D250V + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS887 C173G +R207N + D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS888 C173G + R207N + D250V + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS889 C173G + R207N + D250V + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS890 C173G +R207N + D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS891 C173G + R207N + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS892 C173G + R207N + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS893 C173G + R207N + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS894 C173G +R207N + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS895C173G + R207N + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS896 C173G + R207N + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS897 C173G + R207N + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS898 C173G + R207N + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS899 C173G +R207N + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS900C173G + R207N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS901C173G + D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS902 C173G + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS903 C173G + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS904 C173G + D250V +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS905C173G + D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS906 C173G + D250V + R325A L52F + A108V + R354K and/orG284L/S + H287D + E289A DS907 C173G + D250V + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS908 C173G + D250V + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS909 C173G + D250V L52F +A108V + R354K and/or G284L/S + H287D + E289A DS910 C173G + R325P + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS911 C173G +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS912C173G + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS913C173G + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS914 C173G + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS915 C173G + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS916 C173G + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS917 C173G + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS918 C173G L52F + A108V + R354K and/or G284L/S + H287D +E289A

In a particular embodiment, the variants of the invention comprise theamino acid sequence of SEQ ID NO:2 (or functionally equivalent sequence)and optionally additional amino acid fragments at the C-ter or N-ter. Inanother embodiment, the variants of the invention consist solely on theamino acid sequence of SEQ ID NO:2 (or functionally equivalentsequence). More particularly, in a particular embodiment, the variantsof the invention are deprived of the BRTC-like domain, which correspondsto residues 1 to 129 of SEQ ID NO:1.

According to a second aspect of the invention, the variant of Terminaldeoxynucleotidyl Transferase (TdT) (i) comprises an amino acid sequenceas set forth in SEQ ID NO:2 or a functionally equivalent sequence (suchas, SEQ ID NOs:11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35) oran amino acid sequence having a specified percent sequence identity ofany of the foregoing sequences, with at least three amino acidsubstitutions selected from M63R/Q, L131P, C173G/R, R207L/N, D250V,R325P/N and E328N/L/T/S, or a functionally equivalent residue, whereinthe positions are numbered by reference to the amino acid sequence setforth in SEQ ID NO:1 or as set forth directly elsewhere herein inrespect of their individual SEQ ID NOs, (ii) is able to synthesize anucleic acid fragment without template and (iii) is able to incorporatea modified nucleotide, such as a 3′-O-modified nucleotide, into thenucleic fragment.

For instance, the variant of TdT comprises an amino acid sequence withina specified percent sequence identity of SEQ ID NO:2 and a combinationof substitutions selected from M63R+L131P+R207L, M63R+L131P+R207N,M63R+L131P+D250V, M63R+L131P+R325P, M63R+L131P+R325A, M63R+L131P+E328L,M63R+L131P+E328N, M63R+R207L+D250V, M63R+R207L+R325P, M63R+R207L+R325A,M63R+R207L+E328L, M63R+R207L+E328N, M63R+R207N+D250V, M63R+R207N+R325P,M63R+R207N+R325A, M63R+R207N+E328L, M63R+R207N+E328N, M63R+D250V+R325P,M63R+D250V+R325A, M63R+R325P+E328L, M63R+R325P+E328N, M63R+R325A+E328L,M63R+R325A+E328N, M63Q+L131P+R207L, M63Q+L131P+R207N, M63Q+L131P+D250V,M63Q+L131P+R325P, M63Q+L131P+R325A, M63Q+L131P+E328L, M63Q+L131P+E328N,M63Q+R207L+D250V, M63Q+R207L+R325P, M63Q+R207L+R325A, M63Q+R207L+E328L,M63Q+R207L+E328N, M63Q+D250V+R325P, M63Q+D250V+R325A, M63Q+D250V+E328L,M63Q+D250V+E328N, M63Q+R325P+E328L, M63Q+R325P+E328N, M63Q+R325A+E328L,M63Q+R325A+E328N, L131P+R207L+D250V, L131P+R207L+R325A,L131P+R207L+E328L, L131P+R207L+E328N, L131P+R207N+D250V,L131P+R207N+R325P, L131P+R207N+R325A, L131P+R207N+E328L,L131P+R207N+E328N, L131P+D250V+R325P, L131P+D250V+R325A,L131P+D250V+E328L, L131P+D250V+E328N, L131P+R325P+E328L,L131P+R325P+E328N, L131P+R325A+E328L, L131P+R325A+E328N,R207L+D250V+R325P, R207L+D250V+R325A, R207L+D250V+E328L,R207L+D250V+E328N, R207L+R325P+E328L, R207L+R325P+E328N,R207L+R325A+E328L, R207L+R325A+E328N, R207N+D250V+R325P,R207N+D250V+R325A, R207N+D250V+E328L, R207N+D250V+E328N,R207N+R325P+E328L, R207N+R325P+E328N, R207N+R325A+E328L,R207N+R325A+E328N, D250V+R325P+E328L, D250V+R325P+E328N,D250V+R325A+E328L, D250V+R325A+E328N and R207L+D250V+R325P, orfunctionally equivalent residue(s) wherein the above position numbersare with respect to SEQ ID NO:2.

In a particular embodiment, the variant of TdT comprises an amino acidsequence within a specified percent sequence identity of SEQ ID NO:2, orfunctionally equivalent sequence, with the combination of substitutionsR207L+R325P+E328L (DS928), or functionally equivalent residues.

In a particular embodiment, the variant of TdT comprises an amino acidsequence within a specified percent sequence identity of SEQ ID NO:2, orfunctionally equivalent sequence, with the combination of substitutionsR207N+R325A+E328N (DS950), or functionally equivalent residues.

Such variant may further comprise at least one substitution at positioncorresponding to residues selected from L52, A108, L131, T340, G284,H287, E289, W450, R354 and A510, or functionally equivalent residue(s).

As exposed above, said variant may also comprise the combination ofconstant mutations L52F+A108V+R354K and/or G284L/S+H287D+E289A, orfunctionally equivalent residue(s).

According to a further aspect, the invention provides a variant ofTerminal deoxynucleotidyl Transferase (TdT) which (i) comprises an aminoacid sequence within a specified percent sequence identity of SEQ IDNO:2 or a functionally equivalent sequence, with at least one amino acidsubstitution selected from M63R, M63Q, L131P, R207L, R207N, D250V,R325P, R325A, E328L, E328N, or functionally equivalent residue(s), (ii)is able to synthesize a nucleic acid fragment without a template and(iii) is able to incorporate a 3′-O-modified nucleotide into the nucleicfragment.

In another aspect, the invention provides a variant of Terminaldeoxynucleotidyl Transferase (TdT) which (i) comprises an amino acidsequence within a specified percent sequence identity of SEQ ID NO:2 ora functionally equivalent sequence, with at least the combination ofsubstitutions selected from M63R+L131P, M63R+R207L, M63R+R207N,M63R+D250V, M63R+R325P, M63R+R325A, M63R+E328L, M63R+E328N, M63Q+L131P,M63Q+R207L, M63Q+R207N, M63Q+D250V, M63Q+R325P, M63Q+R325A, M63Q+E328L,M63Q+E328N, L131P+R207L, L131P+R207N, L131P+D250V, L131P+R325P,L131P+R325A, L131P+E328L, L131P+E328N, R207L+D250V, R207L+R325P,R207L+R325A, R207L+E328L, R207L+E328N, R207N+D250V, R207N+R325P,R207N+R325A, R207N+E328L, R207N+E328N, D250V+R325P, D250V+R325A,D250V+E328L, D250V+E328N, R325P+E328L, R325P+E328N, R325A+E328L andR325A+E328N, or functionally equivalent residue(s), wherein thepositions are numbered by reference to the amino acid sequence set forthin SEQ ID NO:2, (ii) is able to synthesize a nucleic acid fragmentwithout a template and (iii) is able to incorporate a 3′-O-modifiednucleotide into the nucleic fragment.

It is thus an object of the invention to provide a TdT variant having anamino acid sequence within a specified percent sequence identity of SEQID NO:2, or functionally equivalent sequence, with any substitution orcombination of substitutions listed in Table 6, listed as “VariableMutations”, or functionally equivalent residue(s) and optionally one orboth combinations of constant mutations L52F+A108V+R354K anG284L/S+H287D+E289A, or functionally equivalent residue(s).

According to a particular embodiment, the variant comprises at least onesubstitution or combination of substitutions as listed in Table 6, andoptionally one or more additional mutation(s).

TABLE 2 Variants of TdT having the amino acid sequence of SEQ ID NO: 2,or an amino acid sequence within a specified percent sequence identitythereof, and further including the following Variable Mutations andOptional Constant Mutations (wherein amino acid position numbers arewith respect to SEQ ID NO: 2). Name Variable Mutations Optional ConstantMutations DS109 M63R + L131P + R207L + D250V + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS110 M63R + L131P +R207L + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS111 M63R + L131P + R207L + D250V + R325P L52F + A108V +R354K and/or G284L/S + H287D + E289A DS112 M63R + L131P + R207L +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS113 M63R + L131P + R207L + D250V + R325A + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS114 M63R + L131P + R207L +D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS115M63R + L131P + R207L + D250V + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS116 M63R + L131P + R207L + D250V + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS117 M63R + L131P +R207L + D250V L52F + A108V + R354K and/or G284L/S + H287D + E289A DS118M63R + L131P + R207L + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS119 M63R + L131P + R207L + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS12O M63R + L131P +R207L + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS121M63R + L131P + R207L + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS122 M63R + L131P + R207L + R325A + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS123 M63R + L131P +R207L + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS124M63R + L131P + R207L + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS125 M63R + L131P + R207L + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS126 M63R + L131P + R207L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS127 M63R + L131P + R207N +D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS128 M63R + L131P + R207N + D250V + R325P + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS129 M63R + L131P + R207N +D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS13OM63R + L131P + R207N + D250V + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS131 M63R + L131P + R207N + D250V + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS132 M63R +L131P + R207N + D250V + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS133 M63R + L131P + R207N + D250V + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS134 M63R + L131P + R207N +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS135M63R + L131P + R207N + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS136 M63R + L131P + R207N + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS137 M63R + L131P + R207N +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS138M63R + L131P + R207N + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS139 M63R + L131P + R207N + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS140 M63R + L131P + R207N +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS141M63R + L131P + R207N + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS142 M63R + L131P + R207N + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS143 M63R + L131P + R207N + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS144 M63R + L131P + R207NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS145 M63R + L131P +D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS146 M63R + L131P + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS147 M63R + L131P + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS148 M63R + L131P +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS149 M63R + L131P + D250V + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS150 M63R + L131P + D250V + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS151 M63R + L131P +D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS152M63R + L131P + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS153 M63R + L131P + D250V L52F + A108V + R354K and/orG284L/S + H287D + E289A DS154 M63R + L131P + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS155 M63R + L131P +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS156M63R + L131P + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289ADS157 M63R + L131P + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS158 M63R + L131P + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS159 M63R + L131P + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS160 M63R + L131P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS161 M63R + L131P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS162 M63R + L131PL52F + A108V + R354K and/or G284L/S + H287D + E289A DS271 M63R + R207L +D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS272 M63R + R207L + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS273 M63R + R207L + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS274 M63R + R207L +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS275 M63R + R207L + D250V + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS276 M63R + R207L + D250V + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS277 M63R + R207L +D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS278M63R + R207L + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS279 M63R + R207L + D250V L52F + A108V + R354K and/orG284L/S + H287D + E289A DS280 M63R + R207L + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS281 M63R + R207L +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS282M63R + R207L + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289ADS283 M63R + R207L + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS284 M63R + R207L + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS285 M63R + R207L + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS286 M63R + R207L + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS287 M63R + R207L + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS288 M63R + R207LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS289 M63R + R207N +D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS290 M63R + R207N + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS291 M63R + R207N + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS292 M63R + R207N +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS293 M63R + R207N + D250V + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS294 M63R + R207N + D250V + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS295 M63R + R207N +D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS296M63R + R207N + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS297 M63R + R207N + D250V L52F + A108V + R354K and/orG284L/S + H287D + E289A DS298 M63R + R207N + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS299 M63R + R207N +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS300M63R + R207N + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289ADS301 M63R + R207N + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS173 M63R + R207N + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS303 M63R + R207N + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS304 M63R + R207N + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS305 M63R + R207N + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS306 M63R + R207NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS307 M63R + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS308M63R + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS309 M63R + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS310 M63R + D250V + R325A + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS311 M63R + D250V +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS312M63R + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289ADS313 M63R + D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS314 M63R + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS315 M63R + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS316 M63R + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS317 M63R + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS318 M63R + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS319 M63R + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS320 M63R + R325A + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS321 M63R + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS322 M63R + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS323 M63R + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS324 M63R L52F + A108V +R354K and/or G284L/S + H287D + E289A DS433 M63Q + L131P + R207L +D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS434 M63Q + L131P + R207L + D250V + R325P + E328N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS435 M63Q + L131P + R207L +D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS436M63Q + L131P + R207L + D250V + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS437 M63Q + L131P + R207L + D250V + R325A +E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS438 M63Q +L131P + R207L + D250V + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS439 M63Q + L131P + R207L + D250V + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS440 M63Q + L131P + R207L +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS441M63Q + L131P + R207L + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS442 M63Q + L131P + R207L + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS443 M63Q + L131P + R207L +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS444M63Q + L131P + R207L + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS445 M63Q + L131P + R207L + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS446 M63Q + L131P + R207L +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS447M63Q + L131P + R207L + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS448 M63Q + L131P + R207L + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS449 M63Q + L131P + R207L + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS450 M63Q + L131P + R207LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS451 M63Q + L131P +R207N + D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS452 M63Q + L131P + R207N + D250V + R325P + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS453 M63Q + L131P +R207N + D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS325 M63Q + L131P + R207N + D250V + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS455 M63Q + L131P + R207N +D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS456 M63Q + L131P + R207N + D250V + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS328 M63Q + L131P + R207N + D250V +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS458 M63Q +L131P + R207N + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS459 M63Q + L131P + R207N + D250V L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS460 M63Q + L131P + R207N + R325P +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS461 M63Q +L131P + R207N + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS462 M63Q + L131P + R207N + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS463 M63Q + L131P + R207N + R325A +E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS464 M63Q +L131P + R207N + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS465 M63Q + L131P + R207N + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS466 M63Q + L131P + R207N + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS467 M63Q + L131P +R207N + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS468M63Q + L131P + R207N L52F + A108V + R354K and/or G284L/S + H287D + E289ADS469 M63Q + L131P + D250V + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS470 M63Q + L131P + D250V + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS471 M63Q + L131P +D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS472M63Q + L131P + D250V + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS473 M63Q + L131P + D250V + R325A + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS474 M63Q + L131P +D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS475M63Q + L131P + D250V + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS476 M63Q + L131P + D250V + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS477 M63Q + L131P + D250V L52F + A108V +R354K and/or G284L/S + H287D + E289A DS478 M63Q + L131P + R325P + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS479 M63Q + L131P +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS354M63Q + L131P + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289ADS481 M63Q + L131P + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS482 M63Q + L131P + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS483 M63Q + L131P + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS484 M63Q + L131P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS485 M63Q + L131P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS486 M63Q + L131PL52F + A108V + R354K and/or G284L/S + H287D + E289A DS595 M63Q + R207L +D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS596 M63Q + R207L + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS597 M63Q + R207L + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS598 M63Q + R207L +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS599 M63Q + R207L + D250V + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS600 M63Q + R207L + D250V + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS601 M63Q + R207L +D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS602M63Q + R207L + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS603 M63Q + R207L + D250V L52F + A108V + R354K and/orG284L/S + H287D + E289A DS604 M63Q + R207L + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS605 M63Q + R207L +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS606M63Q + R207L + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289ADS607 M63Q + R207L + R325A+ E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS608 M63Q + R207L + R325A+ E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS609 M63Q + R207L + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS610 M63Q + R207L + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS611 M63Q + R207L + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS612 M63Q + R207LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS613 M63Q + R207N +D250V + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS614 M63Q + R207N + D250V + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS615 M63Q + R207N + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS616 M63Q + R207N +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS617 M63Q + R207N + D250V + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS618 M63Q + R207N + D250V + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS619 M63Q + R207N +D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS620M63Q + R207N + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS621 M63Q + R207N + D250V L52F + A108V + R354K and/orG284L/S + H287D + E289A DS622 M63Q + R207N + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS623 M63Q + R207N +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS624M63Q + R207N + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289ADS625 M63Q + R207N + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS626 M63Q + R207N + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS627 M63Q + R207N + R325A L52F + A108V +R354K and/or G284L/S + H287D + E289A DS628 M63Q + R207N + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS629 M63Q + R207N + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS630 M63Q + R207NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS631 M63Q + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS632M63Q + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS633 M63Q + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS634 M63Q + D250V + R325A + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS635 M63Q + D250V +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS636M63Q + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289ADS637 M63Q + D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS638 M63Q + D250V + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS639 M63Q + D250V L52F + A108V + R354K and/or G284L/S +H287D + E289A DS640 M63Q + R325P + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS641 M63Q + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS642 M63Q + R325P L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS643 M63Q + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS644 M63Q + R325A + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS645 M63Q + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS646 M63Q + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS647 M63Q + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS648 M63Q L52F + A108V +R354K and/or G284L/S + H287D + E289A DS757 L131P + R207L + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS758L131P + R207L + D250V + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS759 L131P + R207L + D250V + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS760 L131P + R207L +D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS761 L131P + R207L + D250V + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS762 L131P + R207L + D250V + R325AL52F + A108V + R354K and/or G284L/S + H287D + E289A DS763 L131P +R207L + D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS764 L131P + R207L + D250V + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS765 L131P + R207L + D250V L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS766 L131P + R207L + R325P + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS767 L131P +R207L + R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS768 L131P + R207L + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS769 L131P + R207L + R325A + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS770 L131P + R207L + R325A + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS771 L131P +R207L + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS772L131P + R207L + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS773 L131P + R207L + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS774 L131P + R207L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS775 L131P + R207N + D250V + R325P + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS776 L131P + R207N + D250V +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS777L131P + R207N + D250V + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS778 L131P + R207N + D250V + R325A + E328L L52F + A108V +R354K and/or G284L/S + H287D + E289A DS779 L131P + R207N + D250V +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS780L131P + R207N + D250V + R325A L52F + A108V + R354K and/or G284L/S +H287D + E289A DS781 L131P + R207N + D250V + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS782 L131P + R207N + D250V + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS783 L131P +R207N + D250V L52F + A108V + R354K and/or G284L/S + H287D + E289A DS784L131P + R207N + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS785 L131P + R207N + R325P + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS786 L131P + R207N + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS787 L131P + R207N +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS788L131P + R207N + R325A + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS789 L131P + R207N + R325A L52F + A108V + R354K and/orG284L/S + H287D + E289A DS790 L131P + R207N + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS791 L131P + R207N + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS792 L131P + R207N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS793 L131P + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS794L131P + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS795 L131P + D250V + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS796 L131P + D250V + R325A + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS797 L131P + D250V +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS798L131P + D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS799 L131P + D250V + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS800 L131P + D250V + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS801 L131P + D250V L52F + A108V + R354K and/orG284L/S + H287D + E289A DS802 L131P + R325P + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS803 L131P + R325P + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS804 L131P + R325P L52F +A108V + R354K and/or G284L/S + H287D + E289A DS805 L131P + R325A + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS806 L131P +R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS807L131P + R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS808L131P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS809L131P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS810L131P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS921 R207L +D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS922R207L + D250V + R325A + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS923 R207L + D250V + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS924 R207L + D250V + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS925 R207L + D250V + E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS926 R207L +D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS927R207L + D250V L52F + A108V + R354K and/or G284L/S + H287D + E289A DS928R207L + R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS929 R207L + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A DS930 R207L + R325P L52F + A108V + R354K and/or G284L/S +H287D + E289A DS931 R207L + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS932 R207L + R325A + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS933 R207L + R325A L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS934 R207L + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS935 R207L + E328N L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS936 R207L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS937 R207N + D250V + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS938 R207N + D250V +R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS939R207N + D250V + R325P L52F + A108V + R354K and/or G284L/S + H287D +E289A DS940 R207N + D250V + R325A + E328L L52F + A108V + R354K and/orG284L/S + H287D + E289A DS941 R207N + D250V + R325A + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS942 R207N + D250V + R325AL52F + A108V + R354K and/or G284L/S + H287D + E289A DS943 R207N +D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS944R207N + D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS945 R207N + D250V L52F + A108V + R354K and/or G284L/S + H287D +E289A DS946 R207N + R325P + E328L L52F + A108V + R354K and/or G284L/S +H287D + E289A DS947 R207N + R325P + E328N L52F + A108V + R354K and/orG284L/S + H287D + E289A DS948 R207N + R325P L52F + A108V + R354K and/orG284L/S + H287D + E289A DS949 R207N + R325A + E328L L52F + A108V + R354Kand/or G284L/S + H287D + E289A DS950 R207N + R325A + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS951 R207N + R325A L52F +A108V + R354K and/or G284L/S + H287D + E289A DS952 R207N + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS953 R207N + E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS954 R207N L52F + A108V +R354K and/or G284L/S + H287D + E289A DS955 D250V + R325P + E328L L52F +A108V + R354K and/or G284L/S + H287D + E289A DS956 D250V + R325P + E328NL52F + A108V + R354K and/or G284L/S + H287D + E289A DS957 D250V + R325PL52F + A108V + R354K and/or G284L/S + H287D + E289A DS958 D250V +R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS959D250V + R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS960 D250V + R325A L52F + A108V + R354K and/or G284L/S + H287D +E289A DS961 D250V + E328L L52F + A108V + R354K and/or G284L/S + H287D +E289A DS962 D250V + E328N L52F + A108V + R354K and/or G284L/S + H287D +E289A DS963 D250V L52F + A108V + R354K and/or G284L/S + H287D + E289ADS964 R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289ADS965 R325P + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289ADS966 R325P L52F + A108V + R354K and/or G284L/S + H287D + E289A DS967R325A + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS968R325A + E328N L52F + A108V + R354K and/or G284L/S + H287D + E289A DS969R325A L52F + A108V + R354K and/or G284L/S + H287D + E289A DS970 E328LL52F + A108V + R354K and/or G284L/S + H287D + E289A DS971 E328N L52F +A108V + R354K and/or G284L/S + H287D + E289A DS919 R207L + D250V +R325P + E328L L52F + A108V + R354K and/or G284L/S + H287D + E289A DS920R207L + D250V + R325P + E328N L52F + A108V + R354K and/or G284L/S +H287D + E289A

According to some embodiments, a variant of TdT has a substitution orcombination of substitutions described above and has an amino acidsequence within at least 80% identity with SEQ ID NO:2 or with afunctionally equivalent sequence (such as, SEQ ID NOs:11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33 or 35); in some embodiments, such aminoacid sequence is within at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%identity with SEQ ID NO:2 or functionally equivalent sequence (such as,SEQ ID NOs:11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35).

Additional Modifications

In an embodiment, the variant of TdT further includes any type oftagging peptide in its N-terminal, C-terminal or both extremity, such asa His-tag sequence. Said tagging peptide could be used for purification,identification, increasing expression, secretability or increasingcatalytic activity. It will be understood that such different tags areextensively described in the literature and thus all tag known to askilled person are covered by the present invention.

The variants of the invention can also include one or more exogenous orheterologous features at the N- and/or C-terminal regions of the proteinfor use, e.g., in the purification of the recombinant polymerase.

The variant of the invention may further comprise a substitution ofresidues between positions C378 to L406, wherein the positions arenumbered by reference to the amino acid sequence set forth in SEQ IDNO1, or functionally equivalent residues, by residues H363 to C390 ofthe Polμ polymerase of sequence SEQ ID NO:3, wherein the positions arenumbered by reference to the amino acid sequence set forth in SEQ IDNO:3 or functionally equivalent residues.

Advantageously, the variant of TdT comprises at least the amino acidsequence SEQ ID NO:2, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or35, with the disclosed substitution(s) and percent sequence identityvalues.

Nucleic Acids, Expression Cassette, Vector

It is also the purpose of the invention to provide a nucleic acidmolecule encoding a variant of the invention. As used herein, the term“nucleic acid”, “nucleic sequence,” “polynucleotide”, “oligonucleotide”and “nucleotide sequence” are used interchangeably and refer to asequence of deoxyribonucleotides and/or ribonucleotides. In oneembodiment, the nucleic acid is a DNA. In an alternative embodiment, thenucleic acid is RNA. In an alternative embodiment, the nucleic acid isXNA.

The nucleic acids can be in single stranded form or in duplex form or amixture of the two. It can be of recombinant, artificial and/orsynthetic origin and it can comprise modified nucleotides. Suchmodifications could be natural modifications such as epigeneticmodifications, or unnatural modification such as labels, modified bond,a modified purine or pyrimidine base, or a modified sugar. In oneembodiment, nucleic acid molecules are DNA, RNA or XNA bearing naturallyoccurring epigenetic modifications such as methylation,hydfroxymethylation, formylation or 5-carboxylation. In one embodiment,nucleic acid molecules are DNA, RNA or XNA bearing unnaturally occurringmodifications such as fluorescent tag, fluorescent label, interactiongroups.

The nucleic acids of the invention can be in isolated or purified form,and made, isolated and/or manipulated by techniques known per se in theart, e.g., cloning and expression of cDNA libraries, amplification,enzymatic synthesis or recombinant technology. The nucleic acids canalso be synthesized in vitro by well-known chemical synthesistechniques, as described in, e.g., Belousov (1997) Nucleic Acids Res.25:3440-3444.

The invention also encompasses nucleic acids which hybridize, understringent conditions, to a nucleic acid encoding a TdT variant asdefined above. Such stringent conditions include incubations ofhybridization filters at about 42° C. for about 2.5 hours in 2×SSC/0.1%SDS, followed by washing of the filters four times of 15 minutes in1×SSC/0.1% SDS at 65° C. Protocols used are described in such referenceas Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (CurrentProtocols in Molecular Biology (1989)).

The invention also encompasses nucleic acids encoding a TdT variant ofthe invention, wherein the sequence of said nucleic acids, or a portionof said sequence at least, has been engineered using optimized codonusage.

Alternatively, the nucleic acids according to the invention may bededuced from the sequence of the TdT variant according to the inventionand codon usage may be adapted according to the host cell in which thenucleic acids shall be transcribed. These steps may be carried outaccording to methods well known to one skilled in the art and some ofwhich are described in the reference manual Sambrook et al. (Sambrook etal., 2001).

In one embodiment, nucleic acid molecules are polymeric molecules havinglength of more than 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2 000, 3 000, 4 000, 5 000, 6 000, 7 000, 8 000, 9 000, 10 000, 15000, 20 000, 30 000, 40 000, 50 000 or 100 000 nucleotides.

Nucleic acids of the invention may further comprise additionalnucleotide sequences, such as regulatory regions, i.e., promoters,enhancers, silencers, terminators, signal peptides and the like that canbe used to cause or regulate expression of the polypeptide in a selectedhost cell or system.

The present invention further relates to an expression cassettecomprising a nucleic acid according to the invention operably linked toone or more control sequences that direct the expression of said nucleicacid in a suitable host cell. Typically, the expression cassettecomprises, or consists of, a nucleic acid according to the inventionoperably linked to a control sequence such as transcriptional promoterand/or transcription terminator. The control sequence may include apromoter that is recognized by a host cell or an in vitro expressionsystem for expression of a nucleic acid encoding a TdT variant of thepresent invention. The promoter contains transcriptional controlsequences that mediate the expression of the enzyme. The promoter may beany polynucleotide that shows transcriptional activity in the host cellincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell. The control sequence mayalso be a transcription terminator, which is recognized by a host cellto terminate transcription. The terminator is operably linked to the3′-terminus of the nucleic acid encoding the esterase. Any terminatorthat is functional in the host cell may be used in the presentinvention. Typically, the expression cassette comprises, or consists of,a nucleic acid according to the invention operably linked to atranscriptional promoter and a transcription terminator.

The invention also relates to a vector comprising a nucleic acid or anexpression cassette as defined above.

The term “vector” refers to DNA molecule used as a vehicle to transferrecombinant genetic material into a host cell. The major types ofvectors are plasmids, bacteriophages, viruses, cosmids, and artificialchromosomes. The vector itself is generally a DNA sequence that consistsof an insert (a heterologous nucleic acid sequence, transgene) and alarger sequence that serves as the “backbone” of the vector. The purposeof a vector which transfers genetic information to the host is typicallyto isolate, multiply, or express the insert in the target cell. Vectorscalled expression vectors (expression constructs) are specificallyadapted for the expression of the heterologous sequences in the targetcell, and generally have a promoter sequence that drives expression ofthe heterologous sequences encoding a polypeptide. Generally, theregulatory elements that are present in an expression vector include atranscriptional promoter, a ribosome binding site, a terminator, andoptionally present operator. An expression vector can also contain anorigin of replication for autonomous replication in a host cell, aselectable marker, a limited number of useful restriction enzyme sites,and a potential for high copy number. Examples of expression vectors arecloning vectors, modified cloning vectors, specifically designedplasmids and viruses. Expression vectors providing suitable levels ofpolypeptide expression in different hosts are well known in the art. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced.

It is another object of the invention to provide a host cell comprisinga nucleic acid, an expression cassette or a vector as described above.The present invention thus relates to the use of a nucleic acid,expression cassette or vector according to the invention to transform,transfect or transduce a host cell. The choice of the vector willtypically depend on the compatibility of the vector with the host cellinto which it must be introduced.

According to the invention, the host cell may be transformed,transfected or transduced in a transient or stable manner. Theexpression cassette or vector of the invention is introduced into a hostcell so that the cassette or vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector. The term“host cell” also encompasses any progeny of a parent host cell that isnot identical to the parent host cell due to mutations that occur duringreplication. The host cell may be any cell useful in the production of avariant of the present invention, e.g., a prokaryote or a eukaryote. Theprokaryotic host cell may be any Gram-positive or Gram-negativebacterium. The host cell may also be an eukaryotic cell, such as ayeast, fungal, mammalian, insect or plant cell.

The nucleic acid, expression cassette or expression vector according tothe invention may be introduced into the host cell by any method knownby the skilled person, such as electroporation, conjugation,transduction, competent cell transformation, protoplast transformation,protoplast fusion, biolistic “gene gun” transformation, PEG-mediatedtransformation, lipid-assisted transformation or transfection,chemically mediated transfection, lithium acetate-mediatedtransformation, liposome-mediated transformation,

Optionally, more than one copy of a nucleic acid, cassette or vector ofthe present invention may be inserted into a host cell to increaseproduction of the variant.

Modified Nucleotides

According to the invention, the variants of TdT are able to incorporatemodified nucleotides, such as modified 3′O-nucleotides, including3′O-blocked nucleotides.

In the context of the invention, the expression “Modified Nucleotide”refers to a molecule containing a nucleoside (i.e. a base attached to adeoxyribose or ribose sugar molecule) bound to three phosphate groupswhich has at least one additional group on one of its extremity: 2′, 3′,5′ or base. Said additional group blocks further addition of nucleotidesby preventing the formation of any phosphodiester bond(3′O-modification, 2′ or 2′O modifications) or by sterically preventingthe polymerase to attach to any nucleic acid fragments that comprises onits 3′ extremity such modified nucleotide (5′ or base modification).Further, said additional group has advantageously a reversible natureallowing that group to be removed through a specific cleaving reaction.

Nucleosides or nucleotide triphosphates include deoxyadenosinetriphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidinetriphosphate (dCTP) or deoxythymidine triphosphate (dTTP) for examplesof nucleotide containing deoxyribose. Adenosine triphosphate (ATP),guanosine triphosphate (GTP), cytidine triphosphate (CTP) or uridinetriphosphate (UTP) are further examples of nucleotide triphosphatescontaining ribose. Other types of nucleosides may be bound to threephosphates to form nucleotide triphosphates, such as naturally occurringmodified nucleosides and artificial nucleosides.

In a particular embodiment, the modified nucleotide is a 3′O-blockednucleotide, which comprises a group reversibly attached to the 3′ end ofthe nucleotide triphosphate to prevent further nucleotide addition. Saidgroup could have diverse chemical natures, such as azidomethyl, aminoxy,and allyl.

Advantageously, the modified nucleotide is selected from a3′-O—NH2-nucleoside triphosphate, a 3′-O-azidomethyl-nucleosidetriphosphate, a 3′-O-allyl-nucleoside triphosphate, a3′O-(2-nitrobenzyl)-nucleoside triphosphate, or a3′-O-propargyl-nucleoside triphosphate.

In some embodiments, the modified nucleotides comprise a modifiednucleotide or nucleoside molecule comprising a purine or pyrimidine baseand a ribose or deoxyribose sugar moiety having a removable 3′-OHblocking group covalently attached thereto, such that the 3′ carbon atomhas attached a group of the structure:

—O—Z

wherein —Z is any of —C(R′)2-0-R″, —C(R′)2-N(R″)2, —C(R′)2-N(H)R″,—C(R′)2-S—R″ and —C(R′)2-F, wherein each R″ is or is part of a removableprotecting group; each R is independently a hydrogen atom, an alkyl,substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl,heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amidogroup, or a detectable label attached through a linking group; with theproviso that in some embodiments such substituents have up to 10 carbonatoms and/or up to 5 oxygen or nitrogen heteroatoms; or (R′)2 representsan alkylidene group of formula ═C(R′″)2 wherein each R′″ may be the sameor different and is selected from the group comprising hydrogen andhalogen atoms and alkyl groups, with the proviso that in someembodiments the alkyl of each R″ has from 1 to 3 carbon atoms; andwherein the molecule may be reacted to yield an intermediate in whicheach R″ is exchanged for H or, where Z is —(R′)2-F, the F is exchangedfor OH, SH or NH2, preferably OH, which intermediate dissociates underaqueous conditions to afford a molecule with a free 3′-OH; with theproviso that where Z is —C(R′)2-S—R″, both R groups are not H. Incertain embodiments, R′ of the modified nucleotide or nucleoside is analkyl or substituted alkyl, with the proviso that such alkyl orsubstituted alkyl has from 1 to 10 carbon atoms and from 0 to 4 oxygenor nitrogen heteroatoms. In certain embodiments, —Z of the modifiednucleotide or nucleoside is of formula —C(R′)₂—N3. In certainembodiments, Z is an azidomethyl group.

In some embodiments, Z is a cleavable organic moiety with or withoutheteroatoms having a molecular weight of 200 or less. In otherembodiments, Z is a cleavable organic moiety with or without heteroatomshaving a molecular weight of 100 or less. In other embodiments, Z is acleavable organic moiety with or without heteroatoms having a molecularweight of 50 or less.

In a further particular embodiment, “3′O modified nucleotide” refers tonucleotide triphosphate bearing at the 3′ extremity either a3′-O-methyl, 3′-azido, 3′-O-azidomethyl, 3′-O-amino, 3′-aminoxy or3′-O-allyl group. In a further embodiment, the 3′-blocked nucleotidetriphosphate is blocked by either a 3′-O-azidomethyl, 3′-aminoxy or3′-O-allyl group. In other embodiments, “3′O modified nucleotide” refersto nucleotide triphosphate bearing at the 3′ extremity either esters,ethers, carbonitriles, phosphates, carbonates, carbamates,hydroxylamine, borates, nitrates, sugars, phosphoramide,phosphoramidates, phenylsulfenates, sulfates, sulfones or amino acids.In some embodiments, the foregoing 3′-O-blocking groups have a moleculeweight of 100 or less.

In another embodiments, 3′-O-blocking groups of the invention includemethyl, 3′-O-(2-nitrobenzyl), allyl, amine, azidomethyl, tert-butoxyethoxy, or propargyl.

In further particular embodiment, “3′O modified nucleotide” refers to anucleotide triphosphate having a terminator effector modifying groupsuch as those described in WO2016034807.

Interestingly, the variants of the invention exhibit an increasedaffinity for modified nucleotides, as compared to wild type TdT, andthereby an increased ability to incorporate such modified nucleotide ina nucleic acid sequence during nucleic acid synthesis. Moreparticularly, the variants of the invention are able to use andincorporate modified 3′O-nucleotides (and more particularly, 3′O-blockednucleotide) in nucleic acid sequence, which is not possible with wildtype TdT (see Knapp et al. Chem. Eur. J., 2011, 17:2903).

According to a particular aspect, the invention relates to variants ofTdT able to work with modified nucleotides in a nucleic acids enzymaticsynthesis process, particularly with 3′O-modified nucleotides (e.g.,3′O-blocked nucleotide), and having the ability to produce long lengthnucleic acid molecules or derivative of nucleic acid molecules.

Enzymatic Synthesis of Nucleic Acid

It is the purpose of the present invention to provide variants of TdTthat may be used for the synthesis of nucleic acid, such as described inYbert et al, WO2015/159023; Jensen et al, Biochemistry, 57: 1821-1832(2018); Hiatt et al, U.S. Pat. No. 5,808,045. More particularly, it isthe purpose of the present invention to provide variants of TdT suitableto add modified nucleotides to an initiating nucleic acid strand. Theblocking group may be then removed for allowing a new addition ofmodified nucleotide.

According to the invention, by use of a variant of the invention, it ispossible to implement successive cycles comprising additions anddeprotections. This process will therefore allow by multiple cycles ofaddition of a reversible modified nucleotide and further removal of theblocking group to allow the controlled extension of an initiatingnucleic acid strand into a defined sequence.

The present invention contemplates the use of modified TdT according tothe present invention in any enzymatic nucleic acid synthesis process.

It is thus an object of the present invention to provide a method ofsynthesizing a polynucleotide having a predetermined sequence,comprising the steps of:

-   -   a) providing an initiator having a 3′-terminal nucleotide having        a free 3′-hydroxyl;    -   b) repeating cycles of (i) contacting under elongation        conditions the initiator or elongated fragments having free        3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a        TdT variant of the present invention, so that the initiator or        elongated fragments are elongated by incorporation of a        3′-O-blocked nucleoside triphosphate to form 3′-O-blocked        elongated fragments, and (ii) deblocking the elongated fragments        to form elongated fragments having free 3′-hydroxyls, until the        polynucleotide is formed.

It is also the purpose of the present invention to provide a process forsynthesizing a nucleic acid molecule without template, comprising a stepof contacting a nucleic acid primer with both at least one nucleotide,such as at least one 3′O-modified nucleotide, and a variant of theinvention.

The present invention contemplates the concept of enzymatic nucleicacids synthesis process. In such process, nucleic acids molecules are denovo synthesized in absence of any template strand. Accordingly, orderedsequence of nucleotides are coupled to an initiator nucleic acidfragment with the help of the variant of the invention. It will beunderstood that quantitative coupling and more generally high couplingefficiency of each nucleotide to the growing nucleic acid chain is ofgreat importance. It will also be understood that non-terminatornucleotides, such as natural nucleotides or permanent labelednucleotides, will not permit any control over the sequence synthesizedand will result, for example, in uncontrolled and undesiredpoly-additions.

In some embodiments, the method of synthesizing a polynucleotidecomprises the steps of (a) providing an initiator having a free3′-hydroxyl; (b) reacting under extension conditions the initiator or anextension intermediate having a free 3′-hydroxyl with a variant TdT ofthe invention in the presence of a 3′-O-blocked nucleoside triphosphateto produce a 3′-O-blocked extension intermediate; (c) deblocking theextension intermediate to produce an extension intermediate with a free3′-hydroxyl; and (d) repeating steps (b) and (c) until thepolynucleotide is synthesized.

In some embodiments, the method of synthesizing a polynucleotidecomprises the steps of (a) providing an initiator attached to a solidsupport, the intiator being an oligonucleotide having a free3′-hydroxyl; (b) reacting under extension conditions the initiator or anextension intermediate having a free 3′-hydroxyl with a variant TdT ofthe invention in the presence of a 3′-O-blocked nucleoside triphosphateto produce a 3′-O-blocked extension intermediate; (c) washing the solidsupport to remove unincorporated 3′-O-blocked nucleoside triphosphate;(d) deblocking the extension intermediate by exposing the solid supportto a deblocking agent to produce an extension intermediate having a free3′-hydroxyl; and (e) repeating steps (b) and (d) until thepolynucleotide is synthesized. The method may include a further step ofcleaving the completed polynucleotide from the solid support.

In some embodiments, for TdT catalyzed addition reactions, the enzymaticconditions may contain from about 0.20 and about 200 μM of thenucleotide having the removable blocking moiety protecting the3′-hydroxyl and from about 0.20 to 200 μM of free and unmodified3′-hydroxyls derived from the initiating substrate. In some embodiments,the reaction buffer contains from about 10 to about 500 mM potassiumcacodylate buffer (pH between 6.5 and 7.5). and from about 0.01 to about10 mM of a divalent cation (e.g. CoCl₂ or MnCl₂). Other buffercompositions and components may be suitable for particular desiredembodiment of the present invention.

In the context of the invention, the expression “cleaving reaction”refers to any action of substance or physical conditions, which is ableto cleave the additional group previously described on reversiblemodified nucleotides. A person skilled in the art is able to determine acleaving reaction for any previously listed group.

In one embodiment, the cleaving agent is a chemical cleaving agent. Inan alternative embodiment, the cleaving agent is an enzymatic cleavingagent.

It will be understood by the person skilled in the art that theselection of cleaving agent is dependent on the type of 3′-nucleotideblocking group used. For example, tris(2-carboxyethyl)phosphine (TCEP)can be used to cleave a 3′O-azidomethyl groups, palladium complexes canbe used to cleave a 3′O-allyl groups, or sodium nitrite can be used tocleave a 3′O-amino group. In particular embodiment, the cleavingreaction is involving: TCEP, a palladium complex or sodium nitrite.

In particular embodiments, the cleaving reaction is performed in thepresence of additional components such as denaturant (urea, guanidiniumchloride, formamide or betaine for example). In a further embodiment,the cleavage reaction is performed with one or more buffers. It will beunderstood by the person skilled in the art that the choice of buffer isdependent on the exact mechanism of reaction.

The present invention relates to variants of TdT with the capacity toincorporate, in a quantitative way, modified nucleotides. By“quantitative way” or “quantitative reaction”, it is meant a reactionthat goes to completion, i.e. in which reactants are totally convertedinto the product. Polymerase that incorporates in a quantitative wayreversible modified nucleotide is a polymerase able to elongate everyfragment of nucleic acid with all the nucleotides available leading tothe conversion of all the initiating fragments of length n, to fragmentsof length n+1.

As used herein, “initiating fragment” refers to a short oligonucleotidesequence with a free 3′-end, which can be further elongated. In oneembodiment, the initiating fragment is a DNA initiating fragment. In analternative embodiment, the initiating fragment is an RNA initiatingfragment.

In one embodiment, the initiating fragment possesses between 3 and 100nucleotides, in particular between 3 and 20 nucleotides.

In one embodiment, the initiating fragment is single-stranded. In analternative embodiment, the initiating fragment is double-stranded.

In one embodiment, the initiating fragment is immobilized on a solidsupport. The initiating fragment may be attached with various method toa solid support resulting in a stable under the various enzymatic orsynthesis reaction conditions that the fragment will undergo.

In one embodiment, the initiating fragment is immobilized on a solidsupport via a reversible interacting moiety, such as achemically-cleavable linker, an antibody/immunogenic epitope, abiotin/biotin-binding protein or glutathione-GST tag. In a furtherembodiment, the initiating fragment is immobilized on a solid supportvia a chemically-cleavable linker, such as a disulfide, allyl, orazide-masked hemiaminal ether linker.

In an initiating fragment, the immobilized part contains at least onerestriction site. The use of restriction enzymes and restriction sitesto selectively hydrolyze nucleic acids chain at a specific site isdescribe in the literature. Any skilled person will be able to choosethe appropriate restriction enzyme that will match the initiatingfragment cleaving site sequence.

In an alternative embodiment, the initiating fragment contains at leastone uridine. Treatment with uracil-DNA glycosylase (UDG) generates anabasic site. Treatment on an appropriate substrate with anapurinic/apyrimidinic (AP) site endonuclease will extract the nucleicacid strand.

Applications

Described herein is the use of variants of TdT to be used for nucleicacid synthesis, oligonucleotide synthesis, probe synthesis, tagging,nucleic acid amplification, aptamers, therapeutic nucleic acidmolecules, drug target discovery and validation, disease diagnosis,metabolic engineering, data storage, crops improvement, library design,sequencing pools, nucleic acid labeling or attachment or any otherapplication that is involving nucleic acid molecules.

Production of Variant TdTs

Variants of the invention may be produced by mutating known reference orwild type TdT-coding polynucleotides, then expressing it usingconventional molecular biology techniques. For example, the mouse TdTgene (SEQ ID NO:1) may be assembled from synthetic fragments usingconventional molecular biology techniques, e.g. using protocolsdescribed by Stemmer et al, Gene, 164: 49-53 (1995); Kodumal et al,Proc. Natl. Acad. Sci., 101: 15573-15578 (2004); or the like, or it maybe directly cloned from mouse cells using protocols described by Bouleet al, Mol. Biotechnology, 10: 199-208 (1998), or Bentolila et al, EMBOJ., 14: 4221-4229 (1995); or the like.

For example, an isolated TdT gene may be inserted into an expressionvector, such as pET32 (Novagen) to give a vector pCTdT which then may beused to make and express variant TdT proteins using conventionalprotocols. Vectors with the correct sequence may be transformed in E.coli producer strains.

Transformed strains are cultured using conventional techniques topellets from which TdT protein is extracted. For example, previouslyprepared pellets are thawed in 30 to 37° C. water bath. Once fullythawed, pellets are resuspended in lysis buffer composed of 50 mMtris-HCL (Sigma) pH 7.5, 150 mM NaCl (Sigma), 0.5 mM mercaptoethanol(Sigma), 5% glycerol (Sigma), 20 mM imidazole (Sigma) and 1 tab for 100mL of protease cocktail inhibitor (Thermofisher). Careful resuspensionis carried out in order to avoid premature lysis and remaining ofaggregates. Resuspended cells are lysed through several cycles of Frenchpress, until full color homogeneity is obtained. Usual pressure used is14,000 psi. Lysate is then centrifuged for 1 h to 1 h 30 at 10,000 rpm.Centrifugate is pass through a 0.2 μm filter to remove any debris beforecolumn purification.

TdT protein may be purified from the centrifugate in a one-step affinityprocedure. For example, Ni-NTA affinity column (GE Healthcare) is usedto bind the polymerases. Initially the column has been washed andequilibrated with 15 column volumes of 50 mM tris-HCL (Sigma) pH 7.5,150 mM NaCl (Sigma) and 20 mM imidazole (Sigma). Polymerases are boundto the column after equilibration. Then a washing buffer, composed of 50mM tris-HCL (Sigma) pH 7.5, 500 mM NaCl (Sigma) and 20 mM imidazole(Sigma), is applied to the column for 15 column volumes. After wash thepolymerases are eluted with 50 mM tris-HCL (Sigma) pH 7.5, 500 mM NaCl(Sigma) and 0.5M imidazole (Sigma). Fractions corresponding to thehighest concentration of polymerases of interest are collected andpooled in a single sample. The pooled fractions are dialyzed against thedialysis buffer (20 mM Tris-HCl, pH 6.8, 200 mM Na Cl, 50 mM MgOAc, 100mM [NH4]2SO4). The dialysate is subsequently concentrated with the helpof concentration filters (Amicon Ultra-30, Merk Millipore). Concentratedenzyme is distributed in small aliquots, 50% glycerol final is added,and those aliquots are then frozen at −20° C. and stored for long term.5 μL of various fraction of the purified enzymes are analyzed in SDSPAGEgels.

Kits, Enzyme and Nucleotide Composition

A particular aspect of the invention is relative to the composition andthe use of kits comprising a variant of TdT according to the invention,or to any particular aspect of the present invention, with optionallyany combination of one or more components selected from: an initiatingfragment, one or more reversible terminator nucleotides, additionalenzyme and reagents used in a cleaving reaction. Said kits can be usedin a method of enzymatic nucleic acid synthesis.

The present invention covers the composition of matter comprisingvariants of TdT according to the invention, or to any particular aspectof the present invention, with reversible modified nucleotide in a mixwith appropriate buffer and ratio concentration.

EXAMPLES Example 1—Generation, Expression and Purification of Variantsof TdT According to the Invention Expression Strain Generation

The TdT mouse gene has been generated from the pET28 plasmid describedin [Boulé et al., 1998, Mol. Biotechnol. 10, 199-208]. Sequence SEQ IDNo4 (Tag TdT) has been amplified by using the following primers:

(SEQ ID NO: 5) T7-pro: TAATACGACTCACTATAGGG (SEQ ID NO: 6) T7-ter:GCTAGTTATTGCTCAGCGGthrough standard molecular biology techniques. The sequence is thencloned into plasmid pET32 backbone to give the new pCTdT plasmid.

After sequencing pCTdT is transformed into commercial E. coli cells,BL21 (DE3, from Novagen). Growing colonies on plate with kanamycin areisolated and named Ec-CTdT.

Polymerase Variants Generation

The pCTdT vector is used as starting vector. Specific primers comprisingone or several point mutations have been generated from Agilent onlinesoftware (http://www.genomics.agilent.com:80/primerDesignProgram.jsp).The commercially available kit QuickChange II (Agilent) has been used togenerate the desired modified polymerase comprising the targetedmutations. Experimental procedure has followed the supplier's protocol.After generation of the different vectors, each of them have beensequenced. Vectors with the correct sequence have been transformed in E.coli producer strains, as described before. Clones able to grow onkanamycin LB-agar plates are isolated.

Expression

The Ec-CTdT and Ec-DSi or Ec-DSi′ strains have been used for inoculating250 mL erlens with 50 mL of LB media supplemented with appropriateamount of kanamycin. After overnight growth at 37° C., appropriatevolumes of these pre-cultures have been used to inoculate 5 L erlenswith 2L LB media with kanamycin. The initial OD for the 5L cultures ischosen to be 0.01. The erlens are put at 37° C. under strong agitationand the OD of the different cultures are regularly checked. Afterreaching an OD comprised between 0.6 and 0.9 each erlen is supplementedby the addition of 1 mL of 1M IPTG (Isopropylβ-D-1-thiogalactopyranoside, Sigma). The erlens are put back toagitation under a controlled temperature of 37° C. After overnightexpression, the cells are harvested in several pellets. Pelletsexpressing the same variants are pooled and stored at −20° C.,eventually for several months.

Extraction

Previously prepared pellets are thawed in 30 to 37° C. water bath. Oncefully thawed, pellets are resuspended in lysis buffer composed of 50 mMtris-HCL (Sigma) pH 7.5, 150 mM NaCl (Sigma), 0.5 mM mercaptoethanol(Sigma), 5% glycerol (Sigma), 20 mM imidazole (Sigma) and 1 tab for 100mL of protease cocktail inhibitor (Thermofisher). Careful resuspensionis carried out in order to avoid premature lysis and remaining ofaggregates. Resuspended cells are lysed through several cycles of Frenchpress, until full color homogeneity is obtained. Usual pressure used is14,000 psi. Lysate is then centrifuged for 1 h to 1 h 30 at 10,000 rpm.Centrifugate is pass through a 0.2 μm filter to remove any debris beforecolumn purification.

Purification

A one-step affinity procedure is used to purify the produced andextracted polymerase enzymes. A Ni-NTA affinity column (GE Healthcare)is used to bind the polymerases. Initially the column has been washedand equilibrated with 15 column volumes of 50 mM tris-HCL (Sigma) pH7.5, 150 mM NaCl (Sigma) and 20 mM imidazole (Sigma). Polymerases arebound to the column after equilibration. Then a washing buffer, composedof 50 mM tris-HCL (Sigma) pH 7.5, 500 mM NaCl (Sigma) and 20 mMimidazole (Sigma), is applied to the column for 15 column volumes. Afterwash the polymerases are eluted with 50 mM tris-HCL (Sigma) pH 7.5, 500mM NaCl (Sigma) and 0.5M imidazole (Sigma). Fractions corresponding tothe highest concentration of polymerases of interest are collected andpooled in a single sample. The pooled fractions are dialyzed against thedialysis buffer (20 mM Tris-HCl, pH 6.8, 200 mM Na Cl, 50 mM MgOAc, 100mM [NH₄]₂SO₄). The dialysate is subsequently concentrated with the helpof concentration filters (Amicon Ultra-30, Merk Millipore). Concentratedenzyme is distributed in small aliquots, 50% glycerol final is added,and those aliquots are then frozen at −20° C. and stored for long term.5 μL of various fraction of the purified enzymes are analyzed inSDS-PAGE gels.

Results are presented by FIG. 1. The gel shows, for each TdT (bothvariants and wild-type), the column flowthrough (FT) and the differentfractions F1 to F4, corresponding to the elution peaks. A molecularweight marker (M) was also loaded in the gel. FIG. 1 shows that thevariants of TdT according to the invention present a high purity level(about 90%) and a good expression as compared to TdT wild-type (seecolumns F2 and/or F3).

Example 2—Evaluation of the Activity of Variants of TdT with FluorescentPrimers Activity Test

Elongation performance of TdT variants of SEQ ID NO: 2: DS11(M63R+L131P+C173R+R207L+R325P+E328N) DS29(M63R+L131P+C173R+R207N+R325P+E328N), DS173(M63R+C173R+R207L+R325P+E328N), DS659 (L131P+C173R+R207L+R325P+E328N),DS874 (C173G+R207L+R325P+E328L) generated, expressed and purifiedaccording to example 1 is evaluated through the following assay. All theresults are compared with each other and with the wild type TdT enzyme(SEQ ID No 1) and to a control tube lacking any polymerase enzyme.

TABLE 7 Activity test Reagent Concentration Volume H₂O — 12 μL ActivityBuffer 10x 2 μL dNTP 250 μM 2 μL Purified enzyme 20 μM 2 μL Fluorescentprimer DNA 500 nM 2 μL

The Activity buffer comprises, for example, TdT reaction buffer(available from New England Biolabs) supplemented with CoCl₂. Primerused is the following:

(SEQ ID NO: 7) 5′-AAAAAAAAAAAAAAGGGG-3′

The primer has also an ATTO fluorescent dye on the 5′ extremity.

Nucleotides used (noted as dNTP in table 7) are3′-O-amino-2′,3′-dideoxynucleotides-5′-triphosphate (ONH₂, FirebirdBiosciences) such as 3′-O-amino-2′,3′-dideoxyadenosine-5′-triphosphatefor example.

For each different variant tested, one tube is used for the reaction.The reagents are added in the tube, starting from water, and then in theorder of Table 7. After 30 min at 37° C. the reaction is stopped byaddition of formamide (Sigma).

Analysis

The analysis is involving polyacrylamide gel analysis. Samples fromactivity test are analyzed through polyacrylamide 16% (biorad)denaturing gel. Gels are made just before the analysis by pouringpolyacrylamide inside glass plates and let it polymerize. The gel insidethe glass plates is mounted on an adapted tank filed with TBE buffer(Sigma) for the electrophoresis step. The samples to be analyzed areloaded on the top of the gel. A tension of 500 to 2,000V is appliedbetween the top and bottom of the gel for 3 to 6h at room temperature.Once migrated according to the sample target size, system is dismounted,and gel fluorescence is scanned through the use of Typhoon instrument(GE Life Sciences). After image acquisition, ImageJ software(imagej.nih.gov/ij/) is used to analyze the percentage of incorporationof the modified nucleotides.

Results are showed on FIG. 2. For each variant, on the x-axis, theextension percentage has been evaluated as the quantity of expectedelongated product over the total quantity of DNA loaded on the gel. Eachexperiment has been performed in triplicates. The bar height, y-axis,corresponds to the mean value of those three experiments. All thevariants according to the invention show more than a 10-fold increase ofactivity compared to the wt enzyme, confirming the possibility ofdeveloping a nucleic acid synthesis technology with these variants.

Example 3—Evaluation of the Activity of Variants of TdT with UnlabeledPrimer Activity Test

Elongation performance of variants of SEQ ID NO: 2: DS928(R207L+R325P+E328L) and DS950 (R207N+R325A+E328N) generated, expressedand purified according to example 1 was evaluated through the followingassay. All the results are compared with a reference variant (SEQ IDNo9) obtained from previous research and to a control tube lacking anypolymerase enzyme.

TABLE 8 Activity test Reagent Concentration Volume H₂O — 12 μL ActivityBuffer 10x 2 μL dNTP 250 μM 2 μL Purified enzyme 20 μM 2 μL Fluorescentprimer DNA 500 nM 2 μL

Primer used is the following:

(SEQ ID NO: 8) 5′-TTTTTTTTTTTTAAATAAGG-3′

Nucleotides used (noted as dNTP in table 8) were3′-O-amino-2′,3′-dideoxynucleotides-5′-triphosphate (ONH2, FirebirdBiosciences) such as 3′-O-amino-2′,3′-dideoxyadenosine-5′-triphosphatefor example.

For each variant tested one tube was used for the reaction. The reagentswere added in the tube starting from the water and then in the order ofTable 8. After 30 min at 37° C. the reaction was stopped by addition offormamide (Sigma).

Analysis

The analysis used liquid chromatography and mass spectrometer detectionand quantification (LC/MS). Samples from activity test were analyzedthrough LC/MS. Samples were loaded into the LC/MS instrument and astandard oligonucleotide separation method was performed. Acquisition ofdata was followed by deconvolution and spectrum calculation.

Results are showed on FIG. 3. The spectrums correspond to the extensionanalysis of variants DS928, DS950 and references respectively. Initialprimer mass is around 6114 and the expected extended product mass isaround 6447 (emphasized by the arrows). The intensity of the signal(i.e., the height of the peaks) may be directly correlated to thequantity of material. Both variants DS928, DS950 show significantimprovement in the elongation of the starting primer as compared to thereference variant. These results confirm that the new variants accordingto the invention bring indisputable improvement over the TdT of theprior art.

1. A terminal deoxynucleotidyl transferase (TdT) variant comprising an amino acid sequence at least 90% identical to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:29, or SEQ ID NO:31 with an amino acid substitution of cysteine at position 173, wherein the TdT variant (i) is capable of synthesizing a nucleic acid fragment without a template and (ii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.
 2. The TdT variant of claim 1, wherein the modified nucleotide is a 3′-O-modified nucleotide.
 3. The TdT variant of claim 2, wherein the 3′-O-modified nucleotide is a 3′-O—NH₂-nucleoside triphosphate, a 3′-O-azidomethyl-nucleoside triphosphate, a 3′-O-allyl-nucleoside triphosphate, a 3′-(2-nitrobenzyl)-nucleoside triphosphate, or a 3′-O-propargyl-nucleoside triphosphate.
 4. The TdT variant of claim 1, wherein the modified nucleotide is incorporated onto a free 3′-hydroxyl of a nucleic acid fragment.
 5. The TdT variant of claim 1, wherein the TdT variant incorporates the modified nucleotide at a rate greater than that of a wild type TdT.
 6. The TdT variant of claim 1, further comprising a substitution of methionine at position 63 with respect to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:29.
 7. The TdT variant of claim 1, further comprising a substitution of arginine at position 207 with respect to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:29 of SEQ ID NO:31.
 8. The TdT variant of claim 1, further comprising a substitution of arginine at position 208 with respect to SEQ ID NO:21.
 9. The TdT variant of claim 1, further comprising a substitution of arginine at position 324 with respect to SEQ ID NO:11 or SEQ ID NO:13.
 10. The TdT variant of claim 1, further comprising a substitution of arginine at position 331 with respect to SEQ ID NO:17.
 11. The TdT variant of claim 1, further comprising a substitution of arginine at position 325 with respect to SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:31.
 12. The TdT variant of claim 1, further comprising a substitution of arginine at position 328 with respect to SEQ ID NO:
 29. 13. The TdT variant of claim 1, further comprising a substitution of glutamic acid at position 327 with respect to SEQ ID NO:11 or SEQ ID NO:13.
 14. The TdT variant of claim 1, further comprising a substitution of glutamic acid at position 334 with respect to SEQ ID NO: 17 or SEQ ID NO:21.
 15. The TdT variant of claim 1, further comprising a substitution of glutamic acid at position 331 with respect to SEQ ID NO:29.
 16. The TdT variant of claim 1, further comprising a substitution of glutamic acid at position 328 with respect to SEQ ID NO:2, SEQ ID NO:19, or SEQ ID NO:31.
 17. The TdT variant of claim 1, wherein the substitution of the cysteine is G, R, P, A, V, S, N, Q or D.
 18. The TdT variant of claim 6, wherein said substitution of said methionine is R, Q, G, A, V, D, N, H or E.
 19. The TdT variant of claim 7, wherein the substitution of the arginine is N, L, K, H, G, D, A or P.
 20. The TdT variant of claim 13, wherein the substitution of the glutamic acid is N, L, T or S.
 21. The TdT variant of claim 1, wherein the amino acid sequence at least 95% identical to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:29, or SEQ ID NO:31.
 22. The TdT variant of claim 1, wherein the amino acid sequence at least 97% identical to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:29, or SEQ ID NO:31.
 23. A nucleic acid encoding a TdT variant as defined in claim
 1. 24. A method of producing a TdT variant comprising: (a) culturing a host cell comprising a nucleic acid according to claim 21 under conditions suitable to express the nucleic acid encoding the TdT variant; and optionally (b) recovering said TdT variant from the cell culture. 25.-144. (canceled) 